Combined tumor suppressor gene therapy and chemotherapy in the treatment of neoplasms

ABSTRACT

In one embodiment, this invention provides methods of treating mammalian cancer or hyperproliferative cells, said method comprising contacting said cells with a tumor suppressor protein or tumor suppressor nucleic acid and also contacting said cell with at least one adjunctive anti-cancer agent. The invention also provides for a pharmacological composition comprising a tumor suppressor protein or a tumor suppressor nucleic acid and at least one adjunctive anti-cancer agent, and a kit for the treatment of mammalian cancer or hyperproliferative cells.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.10/824,058, filed Apr. 13, 2004, which is a divisional of applicationSer. No. 09/311,772, filed May 13, 1999, which is a continuation ofapplication Ser. No. 09/024,932, filed Feb. 17, 1998, which claimspriority from U.S. Provisional Application No. 60/038,065, filed Feb.18, 1997, now abandoned; and, U.S. Provisional Application No.60/047,834, filed May 28, 1997, now abandoned. Each of theaforementioned applications is explicitly incorporated herein byreference in their entirety and for all purposes.

FIELD OF THE INVENTION

This invention describes novel methods of treating subjects afflictedwith hyperproliferative diseases such as tumors or metastatic disease.In particular, this invention provides methods of inhibiting thehyperproliferation of cells, more specifically neoplastic cells,comprising the combined use of a tumor suppressor gene or gene productand an adjunctive anti-cancer agent.

BACKGROUND OF THE INVENTION

Chromosome abnormalities are often associated with genetic disorders,degenerative diseases, and cancer. In particular, the deletion ormultiplication of copies of whole chromosomes or chromosomal segments,and higher level amplifications of specific regions of the genome arecommon occurrences in cancer. See, for example Smith (1991) BreastCancer Res. Treat., 18: Suppl. 1: 5-14; van de Viler (1991) Became.Beefiest. Acta. 1072: 33-50, Sato (1990) Cancer. Res., 50: 7184-7189. Infact, the amplification of DNA sequences containing proto-oncogenes andthe deletion of DNA sequences containing tumor-suppressor genes, areeach frequently characteristic of tumorigenesis. Dutrillaux (1990)Cancer Genet. Cytogenet., 49: 203-217.

Mutation of the p53 gene is the most common genetic alteration in humancancers (Bartek (1991) Oncogene, 6: 1699-1703, Hollstein (1991) Science,253: 49-53). Moreover, introduction of wild-type p53 in mammalian cancercells lacking endogenous wild-type p53 protein suppresses the neoplasticphenotype of those cells (see, e.g., U.S. Pat. No. 5,532,220).

Of the many available chemotherapeutic drugs, paclitaxel, availablecommercially as TAXOL® (NSC number: 125973) has generated interestbecause of its efficacy in clinical trials against drug-refractorytumors, including ovarian and mammary gland tumors (Hawkins (1992)Oncology, 6: 17-23, Horwitz (1992) Trends Pharmacol. Sci., 13: 134-146,Rowinsky (1990) J. Natl. Canc. Inst., 82: 1247-1259). Recent studies onthe interaction of paclitaxel and tumor suppressor gene therapy showthat reduced levels of tumor suppressor (i.e., p53) correlated withincreased 02/M phase arrest, micronucleation, and p53 independentpaclitaxel-induced apoptosis. In contrast, surviving cells with intactp53 progressed through mitosis and transiently accumulated in thesubsequent G1 phase, coincident with increased p53 and p21^(CiP1.waf1)protein levels (Wahl (1996) Nature Med., 2:72-79). Similarly, Hawkins(1996) Canc. Res., 56: 892-898, showed that inactivation of p53 enhancedsensitivity to certain anti-mitotic agents including paclitaxel. Theauthors suggested that p53 may play a role in DNA repair, therebyallowing cells to progress more readily through S phase even in thepresence of drugs. These studies thus suggest that tumor suppressor genetherapy and drug therapy with antimitotic agents (especially paclitaxeltherapy) act at cross purposes.

BRIEF SUMMARY OF THE INVENTION

This invention provides methods of treating hyperproliferative mammaliancells. The invention is premised, in part, on the surprising discoverythat adjunctive anti-cancer agents in combination with tumor suppressor(e.g., p53) gene therapy provide an enhanced effect in inhibitingproliferation of neoplastic or other cells having deficient tumorsuppressor activity.

Thus, in one embodiment, this invention provides methods of treatingcancer or hyperproliferative cells by contacting the cells with a tumorsuppressor protein or tumor suppressor nucleic acid and with at leastone adjunctive anti-cancer agent. In some embodiments, the methodsinclude co-administration of the tumor suppressor protein or nucleicacid and the adjunctive anti-cancer agent with at least onechemotherapeutic agent. For example, a tumor suppressor nucleic acid(e.g., a nucleic acid encoding p53) can be administered with anadjunctive anti-cancer agent (e.g., paclitaxel) and a DNA damaging agentsuch as cisplatin, carboplatin, navelbine (vinorelbine tartate).

The cancer or hyperproliferative cells are often neoplastic cells. Whenthe cells are present in a tumor the method inhibits tumor growth andthereby provides a method of treating a cancer. Such cancers include,but are not limited to, an ovarian cancer, pancreatic cancer, anon-small cell lung cancer, small cell lung cancer, hepatocarcinoma,melanoma, retinoblastoma, breast tumor, colorectal carcinoma, leukemia,lymphoma, brain tumor, cervical carcinoma, sarcoma, prostate tumor,bladder tumor, tumor of the reticuloendothelial tissues, Wihn's tumor,astrocytoma, glioblastoma, neuroblastoma, ovarian carcinoma,osteosarcoma, renal cancer, or head and neck cancer.

A preferred adjunctive anti-cancer agent is paclitaxel or a paclitaxelderivative while a preferred tumor suppressor nucleic acid is a nucleicacid that encodes a tumor suppressor protein selected from the groupconsisting of p53 protein and its analogues, and a retinoblastoma (RB)protein. A particularly preferred tumor suppressor nucleic acid encodesa wild-type p53 protein and a particularly preferred retinoblastomaprotein is a p110^(RB) or a p56^(RB).

The tumor suppressor nucleic acid is preferably delivered to the targetcell by a vector. Such vectors' viruses have been modified byrecombinant DNA technology to enable the expression of the tumorsuppressor nucleic acid in the target cell. These vectors may be derivedfrom vectors of non-viral (e.g., plasmids) or viral (e.g., adenovirus,adenoassociated virus, retrovirus, herpes virus, vaccinia virus) origin.In the preferred practice of the invention, the vector is arecombinantly modified adenoviral vector. Non-viral vectors arepreferably complexed with agents to facilitate the entry of the DNAacross the cellular membrane. Examples of such non-viral vectorcomplexes include the formulation with polycationic agents whichfacilitate the condensation of the DNA and lipid-based delivery systems.An example of a lipid-based delivery system would include liposome baseddelivery of nucleic acids.

Particularly suitable adenoviral vectors (e.g., for delivery of anucleic acid encoding a wild-type p53 protein) comprise a partial ortotal deletion of a protein I× DNA. In one embodiment, the deletion ofthe protein I× gene sequence extends from about 3500 bp from the 5′viral termini to about 4000 bp from the 5′ viral termini. The vector mayalso comprise a deletion of a non-essential DNA sequence in adenovirusearly region 3 and/or in adenovirus early region 4 and in one embodimentthe deletion is the DNA sequence E1a and/or E1b. A particularlypreferred recombinant adenoviral vector for delivery of a human p53 cDNAcomprises the adenovirus type 2 major late promoter or the human CMVpromoter, and the adenovirus type 2 tripartite leader cDNA. One suchpreferred adenoviral vector is ACN53.

Preferred paclitaxel or paclitaxel derivatives include paclitaxel (soldunder the trademark TAXOL®) and/or TAXOTERE® (docetaxel) with paclitaxel(TAXOL®) being most preferred. Another preferred adjunctive anti-canceris Epothilone. In one particularly preferred embodiment, the tumorsuppressor is A/C/N/53 and the adjunctive anti-cancer agent ispaclitaxel.

The tumor suppressor protein or tumor suppressor nucleic acid can bedispersed in a pharmacologically acceptable excipient. Similarly, theadjunctive anti-cancer (e.g., paclitaxel or paclitaxel derivative) canbe dispersed in a pharmacologically acceptable excipient. The tumorsuppressor protein or tumor suppressor nucleic acid and said paclitaxelor paclitaxel derivative can both be dispersed in a single composition(comprising one or multiple excipient(s)).

The tumor suppressor (protein or nucleic acid) and/or the adjunctiveanti-cancer can be administered intra-arterially, intravenously (e.g.,injected), intraperitoneally and/or intratumorally, together orsequentially. Preferred sites of administration includeintra-hepatic-artery, intraperitoneal, or, where it is desired to treatcells in the head (e.g., neurological cells), into the carotid system ofarteries.

The tumor suppressor protein or nucleic acid can be administered in asingle dose or a multiplicity of treatments, e.g., each separated by atleast about 6 hours, more preferably in least three treatments separatedby about 24 hours.

In another preferred embodiment, the tumor suppressor protein or tumorsuppressor nucleic acid is administered (with or without an adjunctiveanti-cancer agent) in a total dose ranging from about 1×10⁹ to about1×10¹⁴, or about 1×10⁹ to about 7.5×10¹⁵, preferably about 1×10¹¹ toabout 7.5×10¹³, adenovirus particles in a treatment regimen selectedfrom the group consisting of: the total dose in a single dose, the totaldose administered daily over 5 days, the total dose administered dailyover 15 days, and the total dose administered daily over 30 days. Thedose can also be administered continuously for 1 to 30 days. Thepaclitaxel or paclitaxel derivative is administered in a total doseranging from 75-350 mg/m² over 1 hour, 3 hours, 6 hours, or 24 hours ina treatment regimen selected from the group consisting of administrationin a single dose, in the total dose administered daily on each of day 1and day 2, in the total dose administered daily on each of day 1, day 2,and day 3, on a daily dosage for 15 days, on a daily dosage for 30 days,on daily continuous infusion for 15 days, on daily continuous infusionfor 30 days. A preferred dose is 100-250 mg/m² in 24 hours.Alternatively, the paclitaxel or derivative can be administered weeklyat 60 mg/m². This method of administration can be repeated for two ormore cycles (more preferably for three cycles) and the two or morecycles are can be spaced apart by three or four weeks.

In some preferred embodiments, a daily dose in the range of 7.5×10⁹ toabout 7.5×10¹⁵, preferably about 1×10¹² to about 7.5×10¹³, adenovirusparticles can be administered each day for up to 30 days (e.g., aregimen of 2 days or 2 to 5 days to 14 days or 30 days with the samedose being administered each day). The multiple regimen can be repeatedin recurring cycles of 21 to 28 days. Preferred routes of administrationinclude intra-arterial (e.g., intra-hepatic artery), intratumorally, andintraperitoneally.

When the tumor suppressor nucleic acid (e.g., p53) is administered in anadenoviral vector with an adjunctive anti-cancer agent (e.g.,paclitaxel) and a DNA damaging agent (e.g., cisplatin, carboplatin, ornavelbine), the adenoviral vector is administered for 5-14 days at about7.5×10¹² to about 7.5×10¹³ adenoviral particles per day. If theadenoviral vector and paclitaxel is administered with carboplatin, thedose is typically 7.5×10¹³ adenoviral particles per day. For example, adaily dose of about 7.5×10¹² adenoviral particles can be used foradministration to the lung.

This invention also provides for kits for the treatment of mammaliancancer or hyperproliferative cells. The kits include a tumor suppressorprotein or nucleic, acid described herein (more preferably a wild-typep53 protein or nucleic acid (e.g., in a viral or non-viral vector), or aretinoblastoma (RB) protein or nucleic acid); and an adjunctiveanti-cancer agent described herein (e.g., paclitaxel or a paclitaxelderivative) and/or optionally any of the other chemotherapeutic agentsdescribed herein. The kit can optionally further include instructionsdescribing the administration of both the tumor suppressor protein ornucleic acid and the adjunctive anti-cancer agent (and optionally another chemotherapeutic agent) to inhibit the growth or proliferation ofthe cancer or hyperproliferative cells. One particularly preferred kitincludes A/C/N/53 and paclitaxel.

In another embodiment this invention provides pharmacologicalcompositions comprising a tumor suppressor protein or a tumor suppressornucleic acid and an adjunctive anti-cancer agent. In variousembodiments, the pharmacological composition can optionally include anyof the other chemotherapeutic compounds described herein. Oneparticularly preferred composition includes a p53 nucleic acid (e.g.,A/C/N/53) and paclitaxel. The tumor suppressor nucleic acid or proteinand the chemotherapeutic agent (e.g., paclitaxel) can be in differentexcipients or can be contained in a single excipient as describedherein. Where there are multiple excipients, the excipients can beintermixed or held separately (e.g., as in microcapsules).

In still another embodiment, this invention provides a compositioncomprising a mammalian cancer or hyperproliferative cell, wherein saidcell contains an exogenous tumor suppressor nucleic acid or a tumorsuppressor protein. The cell may additionally include an adjunctiveanti-cancer agent such as paclitaxel or a paclitaxel derivative. Theexogenous tumor suppressor nucleic acid or tumor suppressor protein maybe any one or more of the tumor suppressor nucleic acids and/or proteinsdescribed herein. Similarly the cell can be any one or more of thehyperproliferative and/or cancerous cells described herein.

In yet another embodiment, this invention provides a method of treatinga metastatic cell. The method involves contacting the cell with a tumorsuppressor nucleic acid or tumor suppressor polypeptide. Suitable tumorsuppressor nucleic acids or polypeptides include any of the tumorsuppressors nucleic acids and/or polypeptides disclosed herein. Themethod can additionally include contacting the cell with any of theadjunctive anti-cancer agents disclosed herein. In a particularlypreferred embodiment, the method involves topical administration of thetumor suppressor nucleic acid and/or polypeptide to a surgical wound.

In another embodiment, this invention provides particularly preferreddosage regimen. Thus, in one embodiment, this invention provides amethod of treating mammalian cells, where the method involvesadministering to the cells a total dose of a tumor suppressor protein ortumor suppressor nucleic acid, wherein said total dose is administeredin a multiplicity of administrations of incremental doses of said tumorsuppressor protein or tumor suppressor nucleic acid. Preferred multipleadministrations are each separated by at least about 6 hours. Onepreferred administration is in least three treatments separated by about24 hours.

In another embodiment, this invention provides a method of treating amammalian cell. The method involves administering to the cell a totaldose of a tumor suppressor protein or tumor suppressor nucleic acid,wherein the total dose is administered in a multiplicity ofadministrations of incremental doses of tumor suppressor protein ortumor suppressor nucleic acid. The administrations may be spaced by atleast about six hours. The method can involve at least comprising atleast three incremental doses and the doses can be administered daily.In one embodiment, the method can comprise at least three treatmentsseparated by about 24 hours. In another embodiment the method caninvolve tumor administering the tumor suppressor nucleic acid isadministered in a total dose ranging from about 1×10⁹ to about 7.5×10¹⁵,or about 1×10¹¹ to about 7.5×10¹³, adenovirus particles in a treatmentregimen selected from the group consisting of: the total dose in asingle dose, the total dose administered daily over 5 days, the totaldose administered daily over 15 days, and the total dose administereddaily over 30 days. The method may further comprise administeringpaclitaxel or a paclitaxel derivative in a total dose ranging from about75 mg/m² to about 350 mg/m² over 24 hours in a treatment regimenselected from the group consisting of administration in a single dose,in a dose administered daily on day 1 and day 2, in a dose administereddaily on day 1, day 2, and day 3, on a daily dosage for 15 days, on adaily dosage for 30 days, on daily continuous infusion for 15 days, ondaily continuous infusion for 30 days. These treatment regimens may beis repeated for two or more cycles and the two or more cycles can bespaced apart by three or four weeks. The cells thus treated includeneoplastic cells comprising a cancer selected from the group consistingof an ovarian cancer, mesothelioma, pancreatic cancer, a non-small celllung cancer, small cell lung cancer, hepatocarcinoma, melanoma,retinoblastoma, breast tumor, colorectal carcinoma, leukemia, lymphoma,brain tumor, cervical carcinoma, sarcoma, prostate tumor, bladder tumor,tumor of the reticuloendothelial tissues, Wilm's tumor, astrocytoma,glioblastoma, neuroblastoma, osteosarcoma, renal cancer, and head andneck cancer. The treatment treating preferably results in inhibition ofgrowth or proliferation of a tumor as assayed by measurement of thevolume of the tumor.

The invention also provides for a pharmacological composition comprisinga tumor suppressor protein or a tumor suppressor nucleic acid and atleast one adjunctive anti-cancer agent. The adjunctive anti-cancer agentcan be paclitaxel or a paclitaxel derivative. The tumor suppressorprotein or tumor suppressor nucleic acid can be selected from the groupconsisting of a nucleic acid that encodes a wild-type p53 protein, anucleic acid that encodes a retinoblastoma (RB) protein, a wild-type p53protein, and a retinoblastoma (RB) protein.

The retinoblastoma protein can be p110^(RB) or a p56^(RB). The nucleicacid can be contained in a recombinant adenoviral vector. The nucleicacid can be contained in a recombinant adenoviral vector comprising apartial or total deletion of a protein I× DNA and comprising a nucleicacid encoding a P53 protein. In one embodiment, the deletion of theprotein IX gene sequence can extend from about 3500 bp for the 5′ viraltermini to about 4000 bp from the 5′ viral termini. The deletion of DNAcan include sequence designated E1a and E1b. The recombinant adenoviralvector can further comprise the adenovirus type 2 major late promoter orthe human CMV promoter, the adenovirus type 2 tripartite leader cDNA anda human p53 cDNA. In a preferred embodiment, the vector is A/C/N/53. Thecomposition can be paclitaxel, or a paclitaxel derivative or apaclitaxel analogue.

The invention further provides for a composition comprising a mammaliancancer or hyperproliferative cell, wherein said cell contains anexogenous a tumor suppressor nucleic acid or a tumor suppressor proteinand an adjunctive anti-cancer agent. The tumor suppressor nucleic acidcan be a nucleic acid that encodes a tumor suppressor protein selectedfrom the group consisting of wild-type p53 protein, and a retinoblastoma(RB) protein. The retinoblastoma protein can be a p110^(RB) or ap56^(RB). The cells can be present in a mammal. The cells can beneoplastic cells and the neoplastic cells can comprise a cancer selectedfrom the group consisting of an ovarian cancer, pancreatic cancer, anon-small cell lung cancer, small cell lung cancer, hepatocarcinoma,melanoma, retinoblastoma, breast tumor, colorectal carcinoma, leukemia,lymphoma, brain tumor, cervical carcinoma, sarcoma, prostate tumor,bladder tumor, tumor of the reticuloendothelial tissues, Wilm's tumor,astrocytoma, glioblastoma, neuroblastoma, osteosarcoma, renal cancer,and head and neck cancer.

The invention provides for a method of treating a metastatic cell, saidmethod comprising contacting said cell with a tumor suppressor nucleicacid or tumor suppressor polypeptide and an adjunctive anti-canceragent. The contacting can comprise topical administration of a tumorsuppressor nucleic acid to a surgical wound. The method can furtherinclude co-administration of a chemotherapeutic agent, and thechemotherapeutic agent can be cisplatin, carboplatin, or navelbine.

Definitions

The term “adjunctive anti-cancer agent” refers to an agent which has atleast one of the following activities: the ability to modulate ofmicrotubule formation or action, the ability to inhibitpolyprenyl-protein transferase activity, the ability to inhibitangiogenesis, or the ability to inhibit endocrine activity. Adjunctiveanti-cancer agents useful in the invention are described in more detailbelow. As used herein, adjunctive anti-cancer agents of the invention donot include compounds with DNA damaging activity.

“Tumor suppressor genes” are nucleic acids for which loss-of-functionmutations are oncogenic. Thus, the absence, mutation, or disruption ofnormal expression of a tumor suppressor gene in an otherwise healthycell increases the likelihood of, or results in, the cell attaining aneoplastic state. Conversely, when a functional tumor suppressor gene orprotein is present in a cell, its presence suppresses thetumorigenicity, malignancy or hyperproliferative phenotype of the hostcell. Examples of tumor suppressor nucleic acids within this definitioninclude, but are not limited to p110^(RB), p56^(RB), p53, and othertumor suppressors described herein and in copending application U.S.Ser. No. 08/328,673 filed on Oct. 25, 1994. Tumor suppressor nucleicacids include tumor suppressor genes, or nucleic acids derived therefrom(e.g., cDNAs, cRNAs, mRNAs, and subsequences thereof encoding activefragments of the respective tumor suppressor polypeptide), as well asvectors comprising these sequences.

A “tumor suppressor polypeptide or protein” refers to a polypeptidethat, when present in a cell, reduces the tumorigenicity, malignancy, orhyperproliferative phenotype of the cell.

The term “viral particle” refers to an intact virion. The concentrationof infectious adenovirus viral particles is typically determined byspectrophotometric detection of DNA, as described, for instance, byHuyghe (1995) Human Gene Ther., 6:1403-1416.

The terms “neoplasia” or “neoplastic” are intended to describe a cellgrowing and/or dividing at a rate beyond the normal limitations ofgrowth for that cell type.

The term “tumorigenic” or “tumorigenicity” are intended to mean havingthe ability to form tumors or capable of causing tumor formation.

The phrase “treating a cell” refers to the inhibition or amelioration ofone or more disease characteristics of a diseased cell. When used inreference to a cancer cell that is neoplastic (e.g., a mammalian cancercell lacking an endogenous wild-type tumor suppressor protein), thephrase “treating a cell” refers to mitigation or elimination of theneoplastic phenotype. Typically such treatment results in inhibition (areduction or cessation of growth and/or proliferation) of the cell ascompared to the same cell under the same conditions but for thetreatment (e.g., adjunctive anti-cancer agent and or tumor suppressornucleic acid or polypeptide). Such inhibition may include cell death(e.g., apoptosis). These terms when used with reference to a tumor referto inhibition of growth or proliferation of the tumor mass (e.g., asmeasured volumetrically). Such inhibition may be mediated via reductionin growth rate and/or proliferation rate and/or death of cellscomprising the tumor mass. The inhibition of growth or inhibition ofproliferation can be accompanied by an alteration in cellular phenotype(e.g., restoration of morphology characteristic of healthy cells,restoration of contact inhibition, loss of invasive phenotype,inhibition of anchorage independent growth, etc.). For the purposes ofthis disclosure, a diseased cell will have one or more pathologicaltraits. These traits in a diseased cell may include, inter alia,defective expression of one or more tumor suppressor proteins. Defectiveexpression may be characterized by complete loss of one or morefunctional tumor suppressor proteins or a reduction in the level ofexpression of one or more functional tumor suppressor proteins. Suchcells are often neoplastic and/or tumorigenic.

The term “systemic administration” refers to administration of acomposition or drug, such as the recombinant adenoviral vectors of theinvention or the adjunctive anti-cancer or chemotherapeutic compoundsdescribed herein, in a manner that results in the introduction of thecomposition or drug into the circulatory system. The term “regionaladministration” refers to administration of a composition or drug into aspecific anatomical space, such as intraperitoneal, intrathecal,subdural, or to a specific organ, and the like. For example, regionaladministration includes administration of the composition or drug intothe hepatic artery for regional administration to the liver. The term“local administration” refers to administration of a composition or druginto a limited, or circumscribed, anatomic space, such as intratumoralinjection into a tumor mass, subcutaneous injections, intramuscularinjections, and the like. Any one of skill in the art would understandthat local administration or regional administration may also result inentry of the composition or drug into the circulatory system.

The term “reduced tumorigenicity” is used herein to refer to theconversion of hyperproliferative (e.g., neoplastic) cells to a lessproliferative state. In the case of tumor cells, “reducedtumorigenicity” is intended to mean tumor cells that have become lesstumorigenic or non-tumorigenic or non-tumor cells whose ability toconvert into tumor cells is reduced or eliminated. Cells with reducedtumorigenicity either form no tumors in vivo or have an extended lagtime of weeks to months before the appearance of in vivo tumor growth.Cells with reduced tumorigenicity may also result in slower growingthree dimensional tumor mass compared to the same type of cells havingfully inactivated or non-functional tumor suppressor gene growing in thesame physiological milieu (e.g., tissue, organism age, organism sex,time in menstrual cycle, etc.).

As used herein an “active fragment” of a gene or polypeptide includessmaller portion(s) (subsequences) of the gene or nucleic acid derivedtherefrom (e.g., cDNA) that retain the ability to encode proteins havingtumor suppressing activity. Similarly, an active fragment of apolypeptide refers to a subsequence of a polypeptide that has tumorsuppressing protein. One example of an active fragment is p56^(RB) asdescribed, e.g., in copending U.S. Ser. No. 08/328,673 filed on Oct. 25,1994.

The term “malignancy” is intended to describe a tumorigenic cell havingthe ability to metastasize.

“Nucleic acids”, as used herein, may be DNA or RNA. Nucleic acids mayalso include modified nucleotides that permit correct read through by apolymerase and do not alter expression of a polypeptide encoded by thatnucleic acid.

The phrase “nucleotide sequence” includes both the sense and antisensestrands as either individual single strands or in the duplex.

The phrase “DNA sequence” refers to a single or double stranded DNAmolecule composed of the nucleotide bases, adenosine, thymidine,cytosine and guanosine.

The phrase “nucleic acid sequence encoding” refers to a nucleic acidwhich directs the expression of a specific protein or peptide. Thenucleic acid sequences include both the DNA strand sequence that istranscribed into RNA and the RNA sequence that is translated intoprotein. The nucleic acid sequences include both the full length nucleicacid sequences as well as non-full length sequences derived from thefull length sequences. It being further understood that the sequenceincludes the degenerate codons of the native sequence or sequences whichmay be introduced to provide codon preference in a specific host cell.

The phrase “expression cassette”, refers to nucleotide sequences whichare capable of affecting expression of a structural gene in hostscompatible with such sequences. Such cassettes include at leastpromoters and optionally, transcription termination signals. Additionalfactors necessary or helpful in effecting expression may also be used asdescribed herein.

The term “operably linked” as used herein refers to linkage of apromoter upstream from a DNA sequence such that the promoter mediatestranscription of the DNA sequence.

“Isolated” or “substantially pure” when referring to nucleic acidsequences encoding tumor suppressor protein or polypeptide or fragmentsthereof refers to isolated nucleic acids which do not encode proteins orpeptides other than the tumor suppressor protein or polypeptide orfragments thereof.

The term “recombinant” refers to DNA which has been isolated from itsnative or endogenous source and modified either chemically orenzymatically to delete naturally-occurring flanking nucleotides orprovide flanking nucleotides that do not naturally occur. Flankingnucleotides are those nucleotides which are either upstream ordownstream from the described sequence or sub-sequence of nucleotides.

A “vector” comprises a nucleic acid which can infect, transfect,transiently or permanently transduce a cell. It will be recognized thata vector can be a naked nucleic acid, or a nucleic acid complexed withprotein or lipid. The vector optionally comprises viral or bacterialnucleic acids and/or proteins, and/or membranes (e.g., a cell membrane,a viral lipid envelope, etc.). It is recognized that vectors ofteninclude an expression cassette placing the nucleic acid of interestunder the control of a promoter. Vectors include, but are not limited toreplicons (e.g., plasmids, bacteriophages) to which fragments of DNA maybe attached and become replicated. Vectors thus include, but are notlimited to RNA, autonomous self-replicating circular DNA (plasmids), andincludes both the expression and nonexpression plasmids. Where arecombinant microorganism or cell culture is described as hosting an“expression vector” this includes both extrachromosomal circular DNA andDNA that has been incorporated into the host chromosome(s). Where avector is being maintained by a host cell, the vector may either bestably replicated by the cells during mitosis as an autonomousstructure, or is incorporated within the host's genome.

The term effective amount is intended to mean the amount of vector ordrug which achieves a positive outcome on controlling cell growth and/orproliferation.

The abbreviation “C.I.U.” as used herein, stands for “cellularinfectious units.” The C.I.U. is calculated by measuring viral hexonprotein positive cells (e.g., 293 cells) after a 48 hr. infection period(Huyghe (1995) Human Gene Ther. 6:1403-1416).

The abbreviation “m.o.i.” as used herein refers to “multiplicity ofinfection” and is the C.I.U. per cell.

The term “paclitaxel” as used herein refers to the drug commerciallyknown as TAXOL®. TAXOL® inhibits eukaryotic cell replication byenhancing polymerization of tubulin moieties into stabilized microtubulebundles that are unable to reorganize into the proper structures formitosis.

The term “contacting a cell” when referring to contacting with a drugand/or nucleic acid is used herein to refer to contacting in a mannersuch that the drug and/or nucleic acid is internalized into the cell. Inthis context, contacting a cell with a nucleic is equivalent totransfecting a cell with a nucleic acid. Where the drug is lipophilic orthe nucleic acid is complexed with a lipid (e.g., a cationic lipid)simple contacting will result in transport (active, passive and/ordiffusive) into the cell. Alternatively, the drug and/or nucleic acidmay be itself, or in combination with a carrier composition be activelytransported into the cell. Thus, for example, where the nucleic acid ispresent in an infective vector (e.g., an adenovirus) the vector maymediate uptake of the nucleic acid into the cell. The nucleic acid maybe complexed to agents which interact specifically with extracellularreceptors to facilitate delivery of the nucleic acid into the cell,examples include ligand/polycation/DNA complexes as described in U.S.Pat. Nos. 5,166,320 and 5,635,383. Additionally, viral delivery may beenhanced by recombinant modification of the knob or fiber domains of theviral genome to incorporate cell targeting moieties.

The constructs designated herein as “A/C/N/53”, “A/M/N/53”, p110^(RB),p56^(RB), refer to the constructs so designated in copending applicationU.S. Ser. No. 08/328,673, filed on Oct. 25, 1994, InternationalApplication WO 95/11984.

A “conservative substitution”, when describing a protein refers to achange in the amino acid composition of the protein that does notsubstantially alter the protein's activity. Thus, “conservativelymodified variations” of a particular amino acid sequence refers to aminoacid substitutions of those amino acids that are not critical forprotein activity or substitution of amino acids with other amino acidshaving similar properties (e.g., acidic, basic, positively or negativelycharged, polar or non-polar, etc.) such that the substitutions of evencritical amino acids do not substantially alter activity. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. For example, the following six groups each containamino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton (1984) Proteins, W.H. Freeman and Company. Inaddition, individual substitutions, deletions or additions which alter,add or delete a single amino acid or a small percentage of amino acidsin an encoded sequence are also “conservatively modified variations”.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the in vitro inhibition of SK-OV-3 ovarian tumorcells by various concentrations of p53 (A/C/N/53) and/or TAXOL®.

FIG. 2 provides an isobologram analysis for the experiments illustratedin FIG. 1. Synergism between TAXOL® and p53 (A/C/N/53) was observed whenthe cells were pretreated with TAXOL® 24 hours before p53 treatment.

FIGS. 3 a, 3 b, and 3 c illustrate the efficacy of p53 Ad against humanbreast cancer xenografts in nude mice. Mice were given a total dose of2.2×10⁹ C.I.U. adenovirus (A/C/N/53 or Ad) per mouse split into 10injections on days 0-4 and 7-11. Mice were treated with p53 Ad, beta-galAd, or vehicle alone. FIG. 3 a illustrates results with MDA-MB-231tumors. FIG. 3 b illustrates results with MDA-MB-468 (−468) tumors, andFIG. 3 c illustrates results with MDA-MB-435 (−435) tumors.

FIGS. 4 a and 4 b provide p53 Ad (A/C/N/53) dose response curves forMDA-MB-231 (−231) tumors (FIG. 4 a) and for MDA-MB-468 tumors (FIG. 4b). Mice were dosed with 1×10⁷-1×10⁹ C.I.U. p53 Ad (A/C/N/53) split into10 doses administered peritumorally on days 0-4 and 7-11. Averagepercent inhibitions were calculated by comparing the tumor volumes ateach p53 Ad dose with buffer-treated tumors on the days 14/15, 18, 21,24, 28, 30/32, and 35 (MDA-MB-468 tumors only on day 35). The −231tumors averaged 22.5±1.2 mm³ on day 0, while the −468 tumors averaged33.1±1.8 mm³ on day 0.

FIG. 5 provides a comparison of the efficacy of the therapeutic agentwhen administered as a single bolus or as split doses. Tumors(MDA-MB-231) were dosed with a total of 2.2×10⁸ C.I.U. p53 Ad per weekgiven during weeks 1 and 3.

FIG. 6 illustrates the efficacy of multiple cycles of low dose p53 Adagainst large, well-established tumors. A total of 1.32×10⁹ C.I.U. p53Ad was given over 6 weeks to MDA-MB-468 xenografts. (P=plateau incontrol tumor growth rate; E=end of dosing.).

FIGS. 7 a, 7 b, and 7 c illustrate the in vivo inhibition of MDA-MB-468tumors in nude mice administered 1×10⁹ C.I.U. p53 Ad (A/C/N/53) as asingle bolus injection (FIG. 7 a) or split into 3 injections (FIG. 7 b)or 5 injections (FIG. 7 c).

FIG. 8 illustrates of the ability of low dose dexamethasone to suppressthe inhibition of tumor growth mediated by NK cells in scid mice.MDA-MB-231 tumors were dosed with a total of 2×10⁹ C.I.U. beta-gal Ad(1.1×10¹¹ viral particles) split into 10 injections given on days 14-18and 21-25. Subcutaneous dexamethasone (or placebo) pellets released 83.3μg steroid per day.

FIG. 9. Comparison of combination p53 and cisplatin therapy on normaland tumor cells.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides new methods of inhibiting the growth and/orproliferation of cells, more particularly the growth and proliferationof cancer cells. In one embodiment, the methods involve contacting thecells with a tumor suppressor nucleic acid, or tumor suppressor proteinand with an adjunctive anti-cancer agent. Typically, the tumorsuppressor protein or nucleic acid used will be the same species as thetumor suppressor protein that is lacking. Thus, where the cell lacksendogenous p53 activity, a p53 protein or p53 nucleic acid will be used.

It was a surprising discovery of this invention that, contrary to theresults described in previous studies (see, e.g., Wahl et al. (1996)Nature Med., 2(1): 72-79, and Hawkins et al. (1996) Canc. Res. 56:892-898), the treatment of mammalian cells lacking or deficient inendogenous wild-type tumor suppressor protein (i.e., many neoplasticcells), with both an adjunctive anti-cancer agent (e.g., paclitaxel(TAXOL®)) and a tumor suppressor gene or polypeptide (e.g., p53) resultsin inhibition of proliferation and/or growth of the cells greater thanthat observed with either the chemical treatment or the tumor suppressorconstruct alone. Moreover, it was a discovery of this invention thatpretreatment with adjunctive anti-cancer agents dramatically increasesthe anti-proliferative effect of a tumor suppressor nucleic acid.Without being bound by a particular theory, it is believed that possiblemeans by which an adjunctive anti-cancer agent may contribute to thisenhanced effect is: to increase the transfection efficiency of variousgene therapy vectors (e.g., adenovirus vectors); or, to increaseexpression levels of the tumor suppressor gene; or, to stabilizemicrotubules to assist in intracellular virus transport; or, to provideenhanced effect through the interaction of various intracellularmechanisms (e.g., signaling pathways, apoptotic pathways, cell cyclingpathways).

Thus, in one embodiment, this invention provides methods of inhibitingdiseased mammalian cells lacking, or deficient in, an endogenouswild-type tumor suppressor protein of cells by contacting them with anadjunctive anti-cancer agent and with a tumor suppressor nucleic acidand/or tumor suppressor polypeptide. When the cells are present in atumor the method inhibits tumor growth and thereby provides a method oftreating a cancer. Particularly preferred tumor suppressor nucleic acidsor polypeptides include p53, RB, h-NUC (see, e.g., Chen (1995) CellGrowth Differ., 6:199-210) or active fragments thereof (e.g., p110^(RB),p56^(RB)), while particularly preferred adjunctive anti-cancer agents(compounds) include paclitaxel and compounds with paclitaxel-likeactivity such as paclitaxel derivatives (e.g., analogues).

It was also a discovery of this invention that contacting of cells witha tumor suppressor nucleic acid and/or polypeptide can inhibitmetastatic cells. Such inhibition can take the form of inhibition of theformation, growth, migration, or reproduction of metastatic cells. Inone embodiment, the inhibition can be characterized by the inhibition(e.g., reduction and/or elimination) in the appearance of neoplasmsremote from the primary tumor. This invention thus provides methods fortreating (mitigating or eliminating) the progression of metastaticdisease. The methods involve contacting metastatic cells with a tumorsuppressor nucleic acid and/or polypeptide. In a particularly preferredembodiment, this method may involve contacting the cells in a surgicalwound site (e.g., after removal (debulking) of a tumor mass) with atumor suppressor nucleic acid and/or tumor suppressor polypeptide incombination with adjunctive anti-cancer agent. The cells canadditionally be contacted with an adjunctive anti-cancer agent asdescribed herein.

In still another embodiment, this invention provides for advantageoustreatment regimens utilizing tumor suppressor genes and gene products.In part, these treatment regimens are based on the surprising discoverythat tumor suppressor nucleic acids and/or polypeptides are moreeffective in inhibiting cell or tumor growth when delivered in multipleadministrations rather than in a single bolus.

The order in which the tumor suppressor and adjunctive anti-canceragents are administered is not critical to the invention. Thus thecomposition(s) can be administered simultaneously or sequentially. Forinstance, in one embodiment, pretreatment of a cell with at least oneadjunctive anti-cancer agent (alone or in combination with achemotherapeutic agent) increases the efficacy of a subsequentlyadministered tumor suppressor nucleic acid and/or polypeptide. In oneembodiment, the chemotherapeutic agent is administered before theadjunctive anti-cancer agent and the tumor suppressor nucleic acidand/or polypeptide. In another embodiment, the adjunctive anti-canceragent (alone or in combination with a chemotherapeutic agent) isadministered simultaneously with the tumor suppressor nucleic acidand/or polypeptide. In a further embodiment, the tumor suppressornucleic acid and/or polypeptide is administered after the tumorsuppressor nucleic acid and/or polypeptide.

The anti-tumor effect of administering the composition and methods ofthe invention also includes an anti-tumor, non-specific effect, theso-called “bystander effect,” (see, e.g., Zhang (1996) Cancer MetastasisRev., 15:385-401 and Okada (1996) Gene Ther., 3:957-96). Furthermore,the immune system can also be manipulated to selectively accentuate (ordepress) the humoral or the cellular arm of the immune system, i.e.,modulate the B cell and/or T cell (e.g., a cytotoxic lymphocyte (CTL) ortumor infiltrating lymphocyte (TIL)) response. For example, an increasein TILs is observed upon administration of a p53-expressing adenovirusto humans. Specifically, an increase in TILs (phenotypically T helpercells, CD3⁺ and CD4⁺) is observed upon intra-hepatic arterialadministration of a p53-expressing adenovirus for the treatment ofmetastatic hepatic carcinoma, as described in detail below.

It is recognized that the methods of this invention are not restrictedto the use of a single adjunctive anti-cancer agent or even the use of asingle chemotherapeutic. Thus this invention provides for methods ofinhibiting diseased mammalian cells lacking an endogenous tumorsuppressor protein, or a tumor comprising such cells, by contacting thecells or tumor with a tumor suppressor nucleic acid and one or moreadjunctive anti-cancer agent as described herein.

I. Adjunctive Anti-Cancer Agents

A) Microtubule Affecting Agents

As explained above, in one embodiment, this invention provides methodsof inhibiting diseased cells lacking an endogenous tumor suppressorprotein by contacting the cells with a tumor suppressor protein or tumorsuppressor nucleic acid and an adjunctive anti-cancer agent such as amicrotubule affecting agent (e.g., paclitaxel, a paclitaxel derivativeor a paclitaxel-like compound). As used herein, a microtubule affectingagent is a compound that interferes with cellular mitosis, i.e., havingan anti-mitotic effect, by affecting microtubule formation and/oraction. Such agents can be, for instance, microtubule stabilizing agentsor agents which disrupt microtubule formation.

Microtubule affecting agents useful in the invention are well known tothose of skill in the art and include, but are not limited toallocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(TAXOL®, NSC 125973), TAXOL® derivatives (e.g., NSC 608832),thiocolchicine (NSC 361792), trityl cysteine (NSC 83265), vinblastinesulfate (NSC 49842), vincristine sulfate (NSC 67574), epothilone A,epothilone, and discodermolide (see Service, (1996) Science, 274: 2009)estramustine, nocodazole, MAP4, and the like. Examples of such agentsare also described in the scientific and patent literature, see, e.g.,Bulinski (1997) J. Cell Sci. 110:3055-3064; Panda (1997) Proc. Natl.Acad. Sci. USA 94:10560-10564; Muhlradt (1997) Cancer Res. 57:3344-3346;Nicolaou (1997) Nature 387:268-272; Vasquez (1997) Mol. Biol. Cell.8:973-985; Panda (1996) J. Biol. Chem. 271:29807-29812.

Particularly preferred agents are compounds with paclitaxel-likeactivity. These include, but are not limited to paclitaxel andpaclitaxel derivatives (paclitaxel-like compounds) and analogues.Paclitaxel and its derivatives are available commercially. In addition,methods of making paclitaxel and paclitaxel derivatives and analoguesare well known to those of skill in the art (see, e.g., U.S. Pat. Nos.5,569,729; 5,565,478; 5,530,020; 5,527,924; 5,508,447; 5,489,589;5,488,116; 5,484,809; 5,478,854; 5,478,736; 5,475,120; 5,468,769;5,461,169; 5,440,057; 5,422,364; 5,411,984; 5,405,972; and 5,296,506).

Additional microtubule affecting agents can be assessed using one ofmany such assays known in the art, e.g., a semiautomated assay whichmeasures the tubulin-polymerizing activity of paclitaxel analogs incombination with a cellular assay to measure the potential of thesecompounds to block cells in mitosis (see Lopes (1997) Cancer Chemother.Pharmacol., 41:37-47).

Generally, activity of a test compound is determined by contacting acell with that compound and determining whether or not the cell cycle isdisrupted, in particular, through the inhibition of a mitotic event.Such inhibition may be mediated by disruption of the mitotic apparatus,e.g., disruption of normal spindle formation. Cells in which mitosis isinterrupted may be characterized by altered morphology (e.g.,microtubule compaction, increased chromosome number, etc.).

In a preferred embodiment, compounds with possible tubulinpolymerization activity are screened in vitro. In a preferredembodiment, the compounds are screened against cultured WR21 cells(derived from line 69-2 wap-ras mice) for inhibition of proliferationand/or for altered cellular morphology, in particular for microtubulecompaction. In vivo screening of positive-testing compounds can then beperformed using nude mice bearing the WR21 tumor cells. Detailedprotocols for this screening method are described by Porter (1995) Lab.Anim. Sci., 45(2):145-150.

Other methods of screening compounds for desired activity are well knownto those of skill in the art. Typically such assays involve assays forinhibition of microtubule assembly and/or disassembly. Assays formicrotubule assembly are described, for example, by Gaskin et al. (1974)J. Molec. Biol., 89: 737-758. U.S. Pat. No. 5,569,720 also provides invitro and in vivo assays for compounds with paclitaxel-like activity.

B) Polyprenyl-Protein Transferase Inhibitors

In still another embodiment, this invention provides for the combineduse of tumor suppressor nucleic acids and/or polypeptides andpolyprenyl-protein transferase inhibitors. Particularly preferredpolyprenyl-protein transferase inhibitors include, but are not limitedto farnesyl-protein transferase (FPT) inhibitors, geranylgeranyl-proteintransferase inhibitors, and other monoterpene protein transferases.Examples of compounds that are polyprenyl-protein transferase inhibitorsare well known in the scientific and patent literature, see, e.g.,(Zhang (1997) J. Biol. Chem., 272:10232-10239; Njoroge (1997) J. Med.Chem., 40:4290-4301; Mallams (1997) Bioorg. Med. Chem., 5:93-99.

Exemplary compounds that are farnesyl-protein transferase inhibitors aregiven below:

The FPT inhibitor, designated “FPT39,” as described in InternationalApplication WO 97/23478, filed Dec. 19, 1996, where FPT39 is designatedcompound “39.0,” see pg 95 of WO 97/23478.

As described infra, when FPT39 is used in combination therapy with a p53expressing adenovirus of the invention against prostate tumor cells andmammary tumor cells, the combination was more effective at killing tumorcells than either agent alone.

Oligopeptides (mostly tetrapeptides, but also pentapeptides includingthe formula Cys-Xaa1-Xaa2-Xaa3: EPA 461,489; EPA 520,823; EPA 528,486;and WO 95/11917).

Peptido-mimetic compounds, especially Cys-Xaa-Xaa-Xaa mimetics: EPA535,730, EPA 535,731; EPA 618,221; WO 94/09766; WO 94/10138; WO94/07966; U.S. Pat. No. 5,326,773, U.S. Pat. No. 5,340,828; U.S. Pat.No. 5,420,245; WO 95/20396; U.S. Pat. No. 5,439,918; and WO 95/20396.

Farnesylated peptide mimetic compounds—specifically farnesylatedCys-Xaa-Xaa-Xaa mimetic: GB-A2.276,618.

Other peptido-mimetic compounds: U.S. Pat. No. 5,352,705, WO 94/00419;WO 95/00497; WO 95/09000; WO 95/09001; WO 91/12612; WO 95/25086; EPA675,112, and FR-A 2,718,149.

Fused-ring tricyclic benzocycloheptapyridines: WO 95/10514; WO 95/10515;WO 95/10516; WO 96/30363; WO 96/30018; WO 96/30017; WO 96/30362; WO96/31111; WO 96/31478; WO 96/31477; WO 9631505; International PatentApplication No. PCT/US96/19603, WO 97/23478; U.S. application Ser. No.08/728,104, U.S. application Ser. No. 08/712,989, U.S. application Ser.No. 08/713,705, U.S. application Ser. No. 08/713,703; U.S. applicationSer. No. 08/710,225, U.S. application Ser. No. 08/711,925, U.S.application Ser. No. 08/712,924; U.S. application Ser. No. 08/713,323;and U.S. application Ser. No. 08/713,297.

Farnesyl derivatives: EPA 534,546; WO 94/19357; WO 95/08546, EPA537,007; and WO 95/13059.

Natural products and derivatives: WO 94/18157; U.S. Pat. No. 5,430,055;GB-A 2,261,373, GB-A 2,261,374, GB-A 2,261,375; U.S. Pat. No. 5,420,334,U.S. Pat. No. 5,436,263.

Other compounds: WO 94/26723; WO 95/08542; U.S. Pat. No. 5,420,157; WO95/21815; and WO 96/31501.

C) Anti-Angiogenic Compounds

The tumor suppressor proteins or nucleic acids of this invention canalso be administered in conjunction with antiangiogenic compounds.Preferred antiangiogenic compositions inhibit the formation orproliferation of blood vessels, more preferably the formation and/orproliferation of blood vessels to tumors.

Suitable antiangiogenic compositions include, but are not limited toGalardin (GM6001, Glycomed, Inc., Alameda, Calif.), endothelial responseinhibitors (e.g., agents such as interferon alpha, TNP-470, and vascularendothelial growth factor inhibitors), agents that prompt the breakdownof the cellular matrix (e.g., Vitaxin (human LM-609 antibody, Ixsys Co.,San Diego, Calif.; Metastat, CollaGenex, Newtown, Pa.; and MarimastatBB2516, British Biotech), and agents that act directly on vessel growth(e.g., CM-101), which is derived from exotoxin of Group A Streptococcusantigen and binds to new blood vessels inducing an intense hostinflammatory response; and Thalidomide).

Several kinds of steroids have also been noted to exert antiangiogenicactivity. In particular, several reports have indicated thatmedroxyprogesterone acetate (MPA), a synthetic progesterone, potentlyinhibited neovascularization in the rabbit corneal assay (Oikawa (1988)Cancer Lett., 43: 85). A pro-drug of 5FU, 5′-deoxy-5-fluorouridine(5′DFUR), might be also characterized as an antiangiogenic compound,because 5′DFUR is converted to 5-FU by the thymidine phosphorylaseactivity of PD-ECGF/TP. 5′DFUR might be selectively active forPD-ECGF/TP positive tumor cells with high angiogenesis potential. Recentclinical investigations in showed that 5′DFUR is likely to be effectivefor PD-ECGF/TP-positive tumors. It was showed that a dramaticenhancement of antitumor effect of 5′DFUR appeared in PD-ECGF/TPtransfected cells compared with untransfected wild-type cells (Haraguchi(1993) Cancer Res., 53: 5680-5682)). In addition, combined 5′DFUR+MPAcompounds are also effective antiangiogenics (Yayoi (1994) Int J Oncol.,5: 27-32). The combination of the 5′DFUR+MPA might be categorized as acombination of two angiogenesis inhibitors with different spectrums, anendothelial growth factor inhibitor and a protease inhibitor.Furthermore, in in-vivo experiments using DMBA-induced rat mammarycarcinomas, 5′DFUR exhibited a combination effect with AGM-1470(Yamamoto (1995) Oncol Reports 2:793-796).

Another group of antiangiogenic compounds for use in this inventioninclude polysaccharides capable of interfering with the function ofheparin-binding growth factors that promote angiogenesis (e.g., pentosanpolysulfate).

Other modulators of angiogenesis include platelet factor IV, and AGM1470. Still others are derived from natural sources collagenaseinhibitor, vitamin D3-analogues, fumigallin, herbimycin A, andisoflavones.

D) Endocrine Therapy

Endocrine therapy, which is already established and a representativecytostatic treatment, can lead hormone-dependent cells to be quiescentand can reduce tumor cell number in-vivo and can inhibit tumor growth inpatients with hormone-dependent tumors. Such therapies are expected toaugment the effect of tumor suppressors in the treatment ofhyperproliferative cells. Thus, in another embodiment, this inventionprovides, e.g., for the combined use of a tumor suppressor nucleic acidand/or polypeptide and an anti-estrogen, anti-androgen, oranti-progesterone. Endocrine therapeutics are well known to those ofskill in the art are include, but are not limited to tamoxifen,toremifene (see, e.g., U.S. Pat. No. 4,696,949), flutamide, megace, andlupron, see also, e.g., WO 91/00732, WO 93/10741, WO 96/26201, andGauthier et al., J. Med. Chem., 40: 2117-2122 (1997).

E) Delivery of Adjunctive Anti-Cancer Agents: PharmaceuticalCompositions Pharmaceutical Compositions

The adjunctive anti-cancer agents used in the methods of the inventionare typically combined with a pharmaceutically acceptable carrier(excipient) to form a pharmacological composition. The pharmaceuticalcomposition of the invention can comprise one or more adjunctiveanti-cancer agents with or without a tumor suppressor gene orpolypeptide, e.g., p⁵³ or RB.

Pharmaceutically acceptable carriers can contain a physiologicallyacceptable compound that acts, e.g., to stabilize the composition or toincrease or decrease the absorption of the agent and/or pharmaceuticalcomposition. Physiologically acceptable compounds can include, forexample, carbohydrates, such as glucose, sucrose, or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins, compositions that reduce the clearance orhydrolysis of the adjunctive and cancer agents, or excipients or otherstabilizers and/or buffers. Detergents can also used to stabilize thecomposition or to increase or decrease the absorption of thepharmaceutical composition (see infra for exemplary detergents).

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives which areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound depends, for example, onthe route of administration of the adjunctive anti-cancer agent and onthe particular physio-chemical characteristics of the adjunctiveanti-cancer agent.

The compositions for administration will commonly comprise a solution ofthe adjunctive anti-cancer agent dissolved in a pharmaceuticallyacceptable carrier, preferably an aqueous carrier for water-solubleadjunctive anti-cancer agents. A variety of carriers can be used, e.g.,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of adjunctive anti-cancer agentin these formulations can vary widely, and will be selected primarilybased on fluid volumes, viscosities, body weight and the like inaccordance with the particular mode of administration selected and thepatient's needs.

Routes of Delivery

The adjunctive anti-cancer agents used in the methods of the inventionare useful for and can be delivered alone or as pharmaceuticalcompositions (with or without a tumor suppressor, e.g., p53) by anymeans known in the art, e.g., systemically, regionally, or, locally; byintraarterial, intratumoral, intravenous (IV), parenteral, intrapleuralcavity, topical, oral, or local administration, as subcutaneous,intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal,bladder, vaginal, uterine, rectal, nasal mucosa), intratumoral (e.g.,transdermal application or local injection). Particularly preferredmodes of administration include intraarterial injections, especiallywhen it is desired to have a “regional effect,” e.g., to focus on aspecific organ (e.g., brain, liver, spleen, lungs). For example,intra-hepatic artery injection is preferred if the anti-tumor regionaleffect is desired in the liver; or, intra-carotid artery injection,where it is desired to deliver a composition to the brain (e.g., fortreatment of brain tumors), a carotid artery or an-artery of the carotidsystem of arteries (e.g., occipital artery, auricular artery, temporalartery, cerebral artery, maxillary artery, etc.).

Paclitaxel and certain paclitaxel derivatives are only marginallysoluble in aqueous solutions. In a preferred embodiment, thesecompositions are either delivered directly to the tumor locale (e.g., byinjection, canalization, or direct application during a surgicalprocedure) or they are solubilized in an acceptable excipient. Methodsof administering paclitaxel and its derivatives are well known to thoseof skill in the art (see, e.g., U.S. Pat. Nos. 5,583,153, 5,565,478,5,496,804, 45,484,809. Other paclitaxel derivatives are water solubleanalogues and/or prodrugs (see, U.S. Pat. Nos. 5,411,984 and 5,422,364)and are conveniently administered by any of a variety of methods asdescribed above.

The pharmaceutical compositions of this invention are particularlyuseful for topical administration, e.g., in surgical wounds to treatincipient tumors, neoplastic and metastatic cells. and their precursors.In another embodiment, the compositions are useful for parenteraladministration, such as intravenous administration or administrationinto a body cavity or lumen of an organ.

Treatment Regimens

The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include powder,tablets, pills, capsules and lozenges. It is recognized that theadjunctive anti-cancer compounds (e.g., paclitaxel and related compoundsdescribed of, when administered orally, must be protected fromdigestion. This is typically accomplished either by complexing theadjunctive anti-cancer agent with a composition to render it resistantto acidic and enzymatic hydrolysis or by packaging the adjunctiveanti-cancer agent in an appropriately resistant carrier such as aliposome. Means of protecting compounds from digestion are well known inthe art (see, e.g., U.S. Pat. No. 5,391,377 describing lipidcompositions for oral delivery of therapeutic agents).

Dosages for typical chemotherapeutics are well known to those of skillin the art. Moreover, such dosages are typically advisorial in natureand may be adjusted depending on the particular therapeutic context,patient tolerance, etc. Thus, for example, a typical pharmaceuticalcomposition (I, paclitaxel) dosage for intravenous (IV) administrationwould be about 135 mg/m² administered over 1-24 hours (typically at 1,3, or 6 hours, more preferably 3 hours) and more preferably repeatedevery three weeks for 3 to 6 cycles. To decrease the frequency andseverity of hypersensitivity reactions, patients may also receive about20 mg of dexamethasone (Decadron, and others) orally about 12 hours and6 hours before, and about 50 mg of diphenhydramine (BENADRYL®, andothers) plus about 300 mg of cimetidine (TAGAMET®) or 50 mg of rantidine(ZANTAC®) IV 30 to 60 minutes before treatment with paclitaxel.Considerably higher dosages (e.g., ranging up to up to about 350 mg/m²per day may be used, particularly when the drug is administered to asecluded site and not into the blood stream, such as into a body cavityor into a lumen of an organ. Substantially higher dosages are possibleby any selected route, for example, topical administration. Actualmethods for preparing parenterally administrable compositions will beknown or apparent to those skilled in the art and are described in moredetail in such publications as Remington's Pharmaceutical Science, 15thed., Mack Publishing Company, Easton, Pa. (1980) and U.S. Pat. Nos.5,583,153, 5,565,478, 5,496,804, and 5,484,809. Typical doses, e.g., forintraperitoneal administration, will be 20-150 mg/m² weekly, or about250 mg/m² every 3 weeks.

The compositions containing the adjunctive anti-cancer agents can beadministered for therapeutic treatments. In therapeutic applications,compositions are administered to a patient suffering from a diseasecharacterized by hyperproliferation of one or more cell types in anamount sufficient to cure or at least partially arrest the diseaseand/or its complications. An amount adequate to accomplish this isdefined as a “therapeutically effective dose.” Amounts effective forthis use will depend upon the severity of the disease and the generalstate of the patient's health.

Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the adjunctive anti-cancer agents of thisinvention to effectively treat the patient.

II. Tumor Suppressor Genes and Gene Products

A) Preferred Known Tumor Suppressors

As explained above, in one embodiment, this invention provides methodsof inhibiting the growth and/or proliferation of cells by contacting thecells with a tumor suppressor nucleic acid and an adjunctive anti-canceragent (e.g., paclitaxel, a paclitaxel derivative or a paclitaxel-likecompound).

Tumor suppressor genes are well known to those of skill in the art andinclude, but are not limited to RB, p53, APC, FHIT (see, e.g.,Siprashvili (1997) Proc. Natl. Acad. Sci. USA, 94:13771-13776), BRCA1and BRCA2, VHL, WT, DCC, FAP, NF, MEN, E-cadherin, nm23, MMACI, and PTC.The RB or retinoblastoma gene is the prototypical tumor suppressor andhas been well characterized (see, e.g., Bookstein (1990) Science, 247:712-715, Benedict (1980) Cancer Invest., 8: 535-540, Riley (1990) Ann.Rev. Cell Biol., 10-1-29, and Wienberg (1992) Science, 254: 1138-1146.Perhaps the best characterized tumor suppressor is p53 which has beenimplicated in many neoblastomas as well as in the genetic predispositionto the development of diverse tumors in families with Li-Fraumenisyndrome (see, e.g., Wills (1994) Hum. Gene Therap., 5:1079-1088, U.S.Pat. No. 5,532,220, WO 95/289048, and Harris (1996) J. Nat. Canc. Inst.,88 (20): 1442) which describe the cloning expression and use of p53 ingene therapy). Other tumor suppressors include WT (i.e., WT1 at 11p13)gene characteristic of Wilms' tumor (see Call et al. (1990) Cell, 60:60: 509-520, Gessler (1990) Nature, 343: 774-778, and Rose et al. (1990)Cell, 60: 495-508). The tumor suppressor gene called FHIT, for FragileHistidine Triad, was found in a region on chromosome 3 (3p14.2, alsoreported at 3p21) that is known to be particularly prone totranslocations, breaks, and gaps is believed to lead to esophageal,stomach and colon cancers (see, e.g., Ohta et al. (1996) Cell, 84:587-597, GenBank Accession No: U469227). The tumor suppressor genes DCC(18q21) and FAP are associated with colon carcinoma (see, e.g., Hedricket al. (1994) Genes Dev., 8(10): 1174-1183; GenBank Accession No: X76132for DCC, and Wienberg (1992) Science, 254: 1138-1146 for FAP). The NFtumor suppressors (NF1 at 17q11 and NF2 at 22q12) are associated withneurological tumors (e.g., neurofribromatosis for NF1, see, e.g.,Caivthon et al. (1990) Cell, 62: 193-201, Viskochil et al. (1990) Cell,62: 187-192, Wallace et al. (1990) Science, 249: 181-186, and Xug et al.(1990) Cell, 62: 599-608; and Meningioma and schwannoma for NF2). TheMEN tumor suppressor is associated with tumors of the multiple endocrineneoplasia syndrome (see, e.g., Wienberg, Science, 254: 1138-1146, andMarshall (1991) Cell, 64: 313-326). The VHL tumor suppressor isassociated with von Hippel-Landau disease (Latif (1993) Science, 260:1317-1320, GenBank Accession No: L15409). The widely publicized BRCA1and BRCA2 genes are associated with breast cancer (see, e.g., Skolnick(1994) Science, 266: 66-71, GenBank Accession No: U14680 for BRCA1, andTeng (1996) Nature Genet., 13:241-244; GenBank Accession No: U43746)).In addition, the E-cadherin gene is associated with the invasivephenotype of prostate cancer (Umbas (1992) Cancer Res., 52: 5104-5109,Bussemakers (1992) Cancer Res., 52: 2916-2999, GenBank Accession No:272397). The NM23 gene is associated with tumor metastasis (Dooley(1994) Hum. Genet., 93(1): 63-66, GenBank Accession No: X75598). Othertumor suppressors include DPC4 (identified at 18q21) associated withpancreatic cancer, hMLH1 (3p) and hMSH2 (2p) associated with coloncancers, and CDKN2 (16) and (9p) associated with melanoma, pancreaticand esophageal cancers. Finally, the human PTC gene (a homologue of thedrosophila patched (ptc) gene) is associated with nevoid basal cellcarcinoma syndrome (NBCCS) and with somatic basal cell carcinomas (see,e.g., see Hahn et al. (1996) Cell, 85: 841-851). This list of tumorsuppressor genes is neither exhaustive nor intended to be limiting andis meant simply to illustrate the wide variety of known tumorsuppressors.

B) Identification and Screening of Previously Unknown Tumor Suppressors

Methods of identifying or assaying for tumor suppressor genes are wellknown to those of skill in the art. Typically hyperproliferative cellsare screened for gene loss of which, or mutation of which, is associated(correlated) with the hyperproliferative state. The most stringent testfor a gene to qualify as a tumor suppressor gene (TSG) is its ability tosuppress the tumorigenic phenotype of a tumor or of cells derived from atumor. The tumor suppressor nucleic acid is preferably introduced intotumor cells as a cloned cDNA in an appropriate expression vector, or anindividual chromosome harboring a candidate tumor suppressor gene isintroduced into tumor cells by microcell transfer technique.Alternatively, the tumor suppressor gene product (e.g., a tumorsuppressor polypeptide) is introduced into the cell(s) and theproliferation rate of the cells is measured (e.g., by counting cells ormeasuring tumor volume, etc.). Complete or partial inhibition ofproliferation (e.g., decrease of proliferation rate), contactinhibition, loss of invasive phenotype, cell differentiation, andapoptosis, are all indicators of suppression of the tumorigenicphenotype (reduced susceptibility to the neoplastic state).

Methods of screening tumors to identify altered or underexpressednucleic acids are well known to those of skill in the art. Such methodsinclude, but are not limited to subtractive hybridization (see, e.g.,Hampson (1992) Nucleic Acids Res., 20-2899), comparative genomichybridization ((CGH), see, e.g., WO 93/18186, Kallioniemi (1992)Science, 258: 818), and expression monitoring using high density arraysof nucleic acid probes (see, e.g., Lockhart (1996) Nature Biotechnology,14(13): 1675-1680).

C) Preparation of p53 and Other Tumor Suppressors

As indicated above, this invention involves contacting a cell, e.g., invitro, in physiological solution (e.g., blood), in a tissue organ, ororganism with a tumor suppressor nucleic acid or a tumor suppressor geneproduct such as a polypeptide. The tumor suppressor nucleic acid orpolypeptide can be a nucleic acid or polypeptide of any known tumorsuppressor including, but not limited to RB, p53, h-NUC (Chen (1995)supra), APC, FHIT, BRCA1, BRCA2, VHL, WT, DCC, FAP, NF, MEN, E-cadherin,nm23, MMACI, and PTC as described above. In a preferred embodiment, thetumor suppressor is an RB nucleic acid or polypeptide or a p53 nucleicacid or polypeptide or active fragment(s) thereof.

In a most preferred embodiment, the p53 or RB tumor suppressor nucleicacid is present in an expression cassette under control of a promoterthat expresses the tumor suppressor gene or cDNA when it is located inthe target (e.g., tumor) cell. Methods of constructing expressioncassettes and/or vectors encoding tumor suppressor genes are well knownto those of skill in the art as described below.

1. Preparation of Tumor Suppressor Nucleic Acids

DNA encoding the tumor suppressor proteins or protein subsequences ofthis invention may be prepared by any suitable method including, forexample, cloning and restriction of appropriate sequences or directchemical synthesis (e.g., using existing sequence information asindicated above) by methods such as the phosphotriester method of Narang(1979) Meth. Enzymol., 68: 90-99; the phosphodiester method of Brown etal., Meth. Enzymol., 68: 109-151 (1979); the diethylphosphoramiditemethod of Beaucage et al., Tetra. Lett., 22: 1859-1862 (1981); and thesolid support method of U.S. Pat. No. 4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

In one embodiment, tumor suppressor nucleic acids of this invention maybe cloned using DNA amplification methods such as polymerase chainreaction (PCR). Thus, for example, the nucleic acid sequence orsubsequence is PCR amplified, using a sense primer containing onerestriction site (e.g., NdeI) and an antisense primer containing anotherrestriction site (e.g., HindIII). This will produce a nucleic acidencoding the desired tumor suppressor sequence or subsequence and havingterminal restriction sites. This nucleic acid can then be easily ligatedinto a vector containing a nucleic acid encoding the second molecule andhaving the appropriate corresponding restriction sites. Suitable PCRprimers can be determined by one of skill in the art using the publishedsequence information for any particular known tumor suppressor gene,cDNA, or protein. Appropriate restriction sites can also be added to thenucleic acid encoding the tumor suppressor protein or proteinsubsequence by site-directed mutagenesis. The plasmid containing thetumor suppressor sequence or subsequence is cleaved with the appropriaterestriction endonuclease and then ligated into the vector encoding thesecond molecule according to standard methods.

As indicated above, the nucleic acid sequences of many tumor suppressorgenes are known. Thus, for example, the nucleic acid sequence of p53 isfound in Lamb et al. (1986) Mol. Cell Biol., 6: 1379-1385, GenBankAccession No: M13111). Similarly, the nucleic acid sequence of RB isdescribed by Lee et al. (1987) Nature, 329: 642-645 (GenBank AccessionNo: M28419). The nucleic acid sequences of other tumor suppressors areavailable as indicated above in Section II(a). Using the availablesequence information one of ordinary skill in the art can clone thetumor suppressor genes into vectors suitable for practice in thisinvention.

The p53 and RB tumor suppressors are particularly preferred for use inthe methods of this invention. Methods of cloning p53 and RB intovectors suitable for expression of the respective tumor suppressorproteins or for gene therapy applications are well known to those ofskill in the art. Thus, for example, the cloning and use of p53 isdescribed in detail by Wills (1994) supra; in U.S. Pat. No. 5,532,220,in copending U.S. Ser. No. 08/328,673 filed on Oct. 25, 1994, and in WO95/11984. Typically the expression cassette is constructed with thetumor suppressor cDNA operably linked to a promoter, more preferably toa strong promoter (e.g., the Ad2 major late promoter (Ad2 MLP), or thehuman cytomegalovirus immediate early gene promoter (CMV)). In aparticularly preferred embodiment, the promoter is followed by thetripartite leader cDNA and the tumor suppressor cDNA is followed by apolyadenylation site (e.g., the E1b polyadenylation site) (see, e.g.,copending U.S. Ser. No. 08/328,673, WO 95/11984 and Wills (1994) supra).It will be appreciated that various tissue-specific promoters are alsosuitable. Thus, for example, a tyrosinase promoter can be used to targetexpression to melanomas (see, e.g., Siders (1996) Cancer Res.,56:5638-5646). In a particularly preferred embodiment, the tumorsuppressor cDNA is expressed in a vector suitable for gene therapy asdescribed below.

2. Preparation of Tumor Suppressor Protein a) De Novo Chemical Synthesis

Using known sequences of tumor suppressor polypeptides, the tumorsuppressor proteins or subsequences thereof may be synthesized usingstandard chemical peptide synthesis techniques. Where the desiredsubsequences are relatively short (e.g., when a particular antigenicdeterminant is desired) the molecule may be synthesized as a singlecontiguous polypeptide. Where larger molecules are desired, subsequencescan be synthesized separately (in one or more units) and then fused bycondensation of the amino terminus of one molecule with the carboxylterminus of the other molecule thereby forming a peptide bond.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2 Special Methods in Peptide Synthesis, Parta., Merrifield, et al., J. Am. Chem. Soc., 85: 2149-2156 (1963), andStewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill. (1984).

b) Recombinant Expression

In a preferred embodiment, the tumor suppressor proteins or subsequencesthereof, are synthesized using recombinant DNA methodology. Generallythis involves creating a DNA sequence that encodes the fusion protein,placing the DNA in an expression cassette under the control of aparticular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

Methods of cloning the tumor suppressor nucleic acids into a particularvector are described above. The nucleic acid sequences encoding tumorsuppressor proteins or protein subsequences may then be expressed in avariety of host cells, including E. coli, other bacterial hosts, yeast,and various higher eukaryotic cells such as the COS, CHO and HeLa cellslines and myeloma cell lines. As the tumor suppressor proteins aretypically found in eukaryotes, a eukaryote host is preferred. Therecombinant protein gene will be operably linked to appropriateexpression control sequences for each host. For E. coli this includes apromoter such as the T7, trp, or lambda promoters, a ribosome bindingsite and preferably a transcription termination signal. For eukaryoticcells, the control sequences will include a promoter and preferably anenhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc.,and a polyadenylation sequence, and may include splice donor andacceptor sequences.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant tumor suppressor proteins can bepurified according to standard procedures of the art, including ammoniumsulfate precipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc.N.Y. (1990)). Substantially pure compositions of at least about 90 to95% homogeneity are preferred, and 98 to 99% or more homogeneity aremost preferred. Once purified, partially or to homogeneity as desired,the polypeptides may then be used (e.g., as immunogens for antibodyproduction).

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the tumor suppressor protein(s)may possess a conformation substantially different than the nativeconformations of the constituent polypeptides. In this case, it may benecessary to denature and reduce the polypeptide and then to cause thepolypeptide to re-fold into the preferred conformation. Methods ofreducing and denaturing proteins and inducing re-folding are well knownto those of skill in the art (see, Debinski (1993) J. Biol. Chem., 268:14065-14070; Kreitman (1993) Bioconjug. Chem., 4: 581-585; and Buchner(1992) Anal. Biochem., 205: 263-270). Debinski (1993) supra, forexample, describes the denaturation and reduction of inclusion bodyproteins in guanidine-DTE. The protein is then refolded in a redoxbuffer containing oxidized glutathione and L-arginine.

One of skill will appreciate that many conservative variations of thenucleic acid and polypeptide sequences described herein yieldfunctionally identical products. For example, due to the degeneracy ofthe genetic code, “silent substitutions” (i.e., substitutions of anucleic acid sequence which do not result in an alteration in an encodedpolypeptide) are an implied feature of every nucleic acid sequence whichencodes an amino acid. Similarly, “conservative amino acidsubstitutions,” in one or a few amino acids in an amino acid sequenceare substituted with different amino acids with highly similarproperties (see, the definitions section, supra), are also readilyidentified as being highly similar to a disclosed amino acid sequence,or to a disclosed nucleic acid sequence which encodes an amino acid.Such conservatively substituted variations of each explicitly describedsequence are a feature of the present invention.

One of skill would recognize that modifications can be made to the tumorsuppressor proteins without diminishing their biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification sequences.

Modifications to nucleic acids and polypeptides may be evaluated byroutine screening techniques in suitable assays for the desiredcharacteristic. For instance, changes in the immunological character ofa polypeptide can be detected by an appropriate immunological assay.Modifications of other properties such as nucleic acid hybridization toa target nucleic acid, redox or thermal stability of a protein,hydrophobicity, susceptibility to proteolysis, or the tendency toaggregate are all assayed according to standard techniques.

D) Delivery of Tumor Suppressors to Target Cells

The tumor suppressors used in the methods of this invention can beintroduced to the cells either as a protein or as a nucleic acid. Wherethe tumor suppressor is provided as a protein, a tumor suppressor geneexpression product (e.g., a p53 or an RB polypeptide or fragment thereofpossessing tumor suppressor activity) is delivered to the target cellusing standard methods for protein delivery (see discussion, below).Alternatively, where the tumor suppressor is a tumor suppressor nucleicacid (e.g., a gene, a cDNA, an mRNA, etc.), the nucleic acid isintroduced into the cell using conventional methods of deliveringnucleic acids to cells. These methods typically involve delivery methodsof in vivo or ex vivo gene therapy as described below. Particularlypreferred methods of delivering p53 or RB include lipid or liposomedelivery and/or the use of retroviral or adenoviral vectors.

1. In Vivo Gene Therapy.

In a more preferred embodiment, the tumor suppressor nucleic acids(e.g., cDNA(s) encoding the tumor suppressor protein) are cloned intogene therapy vectors that are competent to transfect cells (such ashuman or other mammalian cells) in vitro and/or in vivo.

Several approaches for introducing nucleic acids into cells in vivo, exvivo and in vitro have been used. These include lipid or liposome basedgene delivery (WO 96/18372; WO 93/24640; Mannino (1988) BioTechniques,6(7): 682-691; Rose, U.S. Pat. No. 5,279,833; WO 91/06309; and Feigner(1987) Proc. Natl. Acad. Sci. USA, 84: 7413-7414) andreplication-defective retroviral vectors harboring a therapeuticpolynucleotide sequence as part of the retroviral genome (see, e.g.,Miller (1990) Mol. Cell. Biol., 10:4239 (1990); Kolberg (1992) J. NIHRes., 4: 43, and Cornetta (1991) Hum. Gene Ther., 2: 215).

For a review of gene therapy procedures, see, e.g., Zhang (1996) CancerMetastasis Rev., 15:385-401; Anderson, Science (1992) 256: 808-813;Nabel (1993) TIBTECH, 11: 211-217; Mitani (1993) TIBTECH, 11: 162-166;Mulligan (1993) Science, 926-932; Dillon (1993) TIBTECH, 11: 167-175;Miller (1992) Nature, 357: 455-460; Van Brunt (1988) Biotechnology,6(10), 149-1154; Vigne (1995) Restorative Neurology and Neuroscience, 8:35-36; Kremer (1995) British Medical Bulletin, 51(1): 31-44; Haddada(1995) Current Topics in Microbiology and Immunology; Doerfler and Böhm(eds) Springer-Verlag, Heidelberg Germany; and Yu (1994) Gene Therapy,1:13-26.

The vectors useful in the practice of the present invention aretypically derived from viral genomes. Vectors which may be employedinclude recombinantly modified enveloped or non-enveloped DNA and RNAviruses, preferably selected from baculoviridiae, parvoviridiae,picornoviridiae, herpesviridiae, poxyiridae, adenoviridiae, orpicomnaviridiae. Chimeric vectors may also be employed which exploitadvantageous ments of each of the parent vector properties (see, e.g.,Feng (1997) Nature Biotechnology, 15:866-870). Such viral genomes may bemodified by recombinant DNA techniques to include the tumor suppressorgene and may be engineered to be replication deficient, conditionallyreplicating or replication competent. In the preferred practice of theinvention, the vectors are replication deficient or conditionallyreplicating. Preferred vectors are derived from the adenoviral,adeno-associated viral and retroviral genomes. In the most preferredpractice of the invention, the vectors are replication incompetentvectors derived from the human adenovirus genome.

Conditionally replicating viral vectors are used to achieve selectiveexpression in particular cell types while avoiding untoward broadspectrum infection. Examples of conditionally replicating vectors aredescribed in Bischoff, et al. (1996) Science, 274:373-376; Pennisi, E.(1996) Science, 274:342-343; Russell, S. J. (1994) Eur. J. of Cancer,30A(8): 1165-1171. Additionally, the viral genome may be modified toinclude inducible promoters which achieve replication or expression ofthe transgene only under certain conditions. Examples of induciblepromoters are known in the scientific literature (see, e.g., Yoshida andHamada (1997) Biochem. Biophys. Res. Comm., 230:426-430; Iida, et al.(1996) J. Virol., 70(9):6054-6059; Hwang, et al. (1997) J. Virol.,71(9):7128-7131; Lee, et al. (1997) Mel. Cell. Biol., 17(9):5097-5105;and Dreher, et al. (1997) J. Biol. Chem., 272(46); 29364-29371. Thetransgene may also be under control of a tissue specific promoter regionallowing expression of the transgene only in particular cell types.

Widely used retroviral vectors include those based upon murine leukemiavirus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immune deficiencyvirus (SIV), human immune deficiency virus (HIV), and combinationsthereof (see, e.g., Buchscher (1992) J. Virol., 66(5) 2731-2739; Johann(1992) J. Virol., 66(5):1635-1640 (1992); Sommerfelt (1990) Virol.,176:58-59; Wilson (1989) J. Viral., 63:2374-2378; Miller (1991) J.Virol., 65:2220-2224; Wong-Staal et al., PCT/US94/05700, and Rosenburgand Fauci (1993) Fundamental Immunology, Third Edition Paul (ed.) RavenPress, Ltd., New York and the references therein, and Yu (1994) supra).The vectors are optionally pseudotyped to extend the host range of thevector to cells which are not infected by the retrovirus correspondingto the vector. The vesicular stomatitis virus envelope glycoprotein(VSV-G) has been used to construct VSV-G-pseudotyped HIV vectors whichcan infect hematopoietic stem cells (Naldini et al. (1996) Science,272:263, and Akkina (1996) J. Virol., 70:2581).

Adeno-associated virus (AAV)-based vectors are also used to transducecells with target nucleic acids, e.g., in the in vitro production ofnucleic acids and peptides, and in in vivo and ex vivo gene therapyprocedures. See, Okada (1996) Gene Ther., 3:957-964; West (1987)Virology 160:38-47; Carter (1989) U.S. Pat. No. 4,797,368; Carter etal., WO 93/24641 (1993); Kotin (1994) Human Gene Therapy, 5:793-801;Muzyczka (1994) J. Clin. Invst., 94:1351, for an overview of AAVvectors. Construction of recombinant AAV vectors are described in anumber of publications, including Lebkowski, U.S. Pat. No. 5,173,414;Tratschin (1985) Mol. Cell. Biol., 5(11):3251-3260; Tratschin (1984)Mol. Cell. Biol., 4: 2072-2081; Hermonat (1984) Proc. Matt. Acad. Sci.USA, 81: 6466-6470; McLaughlin (1988) and Samulski (1989) J. Virol.,63:03822-3828. Cell lines that can be transformed by rAAV include thosedescribed in Lebkowski (1988) Mal. Cell. Biol., 8:3988-3996. Othersuitable viral vectors include herpes virus and vaccinia virus.

In a particularly preferred embodiment, the tumor suppressor gene isexpressed in an adenoviral vector suitable for gene therapy. The use ofadenoviral vectors in vivo, and for gene therapy, is well described inthe patent and scientific literature, e.g., see, Hermens (1997) J.Neurosci. Methods., Jan., 71(1): 85-98; Zeiger (1996) Surgery,120:921-925; Channon (1996) Cardiovasc Res., 32:962-972; Huang (1996)Gene Ther., 3:980-987; Zepeda (1996) Gene Ther., 3:973-979; Yang (1996)Hum. Mol. Genet., 5:1703-1712; Caruso (1996) Proc. Natl. Acad. Sci. USA,93:11302-11306; Rothman (1996) Gene Ther., 3:919-926; Haecker (1996)Hum. Gene Ther., 7:1907-1914. The use of adenoviral vectors is describedin detail in WO 96/25507. Particularly preferred adenoviral vectors aredescribed by Wills (1994) supra; in copending U.S. Ser. No. 08/328,673,and WO 95/11984.

Particularly preferred adenoviral vectors include a deletion of some orall of the protein IX gene. In one embodiment, the adenoviral vectorsinclude deletions of the E1a and/or E1b sequences. In a most preferredembodiment, the adenoviral construct is a p53 encoding construct such asA/C/N/53 or A/M/N/53 (see, e.g., U.S. Ser. No. 08/328,673, and WO95/11984).

Also preferred are vectors derived from the human adenovirus type 2 ortype 5. Such vectors are preferably are replication deficient bymodifications or deletions in the E1a and/or E1b coding regions. Othermodifications to the viral genome to achieve particular expressioncharacteristics or permit repeat administration or lower immune responseare preferred. More preferred are recombinant adenoviral vectors havingcomplete or partial deletions of the E4 coding region, optionallyretaining E4 ORF6 and ORF 6/7. The E3 coding sequence may be deleted butis preferably retained. In particular, it is preferred that the promoteroperator region of E3 be modified to increase expression of E3 toachieve a more favorable immunological profile for the therapeuticvectors. Most preferred are human adenoviral type 5 vectors containing aDNA sequence encoding p53 under control of the cytomegalovirus promoterregion and the tripartite leader sequence having E3 under control of theCMV promoter and deletion of E4 coding regions while retaining E4 ORF6and ORF 6/7. In the most preferred practice of the invention asexemplified herein, the vector is ACN53.

In a particularly preferred embodiment, the tumor suppressor gene is p53or RB. As explained above, the cloning and use of p53 is described indetail by Wills (1994) supra, in copending U.S. Ser. No. 08/328,673filed on Oct. 25, 1994, and in WO 95/11984.

2. Ex Vivo Gene Therapy

In one embodiment, the methods of this invention are used to inhibithyperproliferative (e.g., neoplastic) cells in a subject (e.g., a mammalincluding but not limited to rat, murine, bovine, porcine, equine,canine, feline, largomorph, or human). Pathologic hyperproliferativecells are characteristic of disease states including, but not limited toGrave's disease, psoriasis, benign prostatic hypertrophy, Li-Fraumenisyndrome, breast cancer, sarcomas, bladder cancer, colon cancer, lungcancer, various leukemia and lymphomas and other neoplasms.

Ex vivo application of the methods of this invention, in particular,provide means for depleting a suitable sample of pathologichyperproliferative cells. Thus, for example hyperproliferative cellscontaminating hematopoietic precursors during bone marrow reconstitutioncan be eliminated by the ex vivo application of the methods of thisinvention. Typically such methods involve obtaining a sample from thesubject organism. The sample is typically a heterogenous cellpreparation containing both phenotypically normal and pathogenic(hyperproliferative) cells. The sample is contacted with the tumorsuppressor nucleic acids or proteins and the adjunctive anti-canceragent according to the methods of this invention. The tumor suppressorgene can be delivered, e.g., in a viral vector, such as a retroviralvector or an adenoviral vector. The treatment reduces the proliferationof the pathogenic cells thereby providing a sample containing a higherratio of normal to pathogenic cells which can be reintroduced into thesubject organism.

Ex vivo cell transformation for diagnostics, research, or for genetherapy (e.g., via re-infusion of the transformed cells into the hostorganism) is well known to those of skill in the art. In a preferredembodiment, cells are isolated from the subject organism, transfectedwith the tumor suppressor gene or cDNA of this invention, and re-infusedback into the subject organism (e.g., patient). Various cell typessuitable for ex vivo transformation are well known to those of skill inthe art. Particular preferred cells are progenitor or stem cells (see,e.g., Freshney (1994) Culture of Animal Cells, a Manual of BasicTechnique, third edition, Wiley-Liss, New York, and the references citedtherein for a discussion of how to isolate and culture cells frompatients). Transformed cells are cultured by means well known in theart. See also Kuchler (1977) Biochemical Methods in Cell Culture andVirology, Kuchler, R. J., Dowden, Hutchinson and Ross, Inc., and Atlas(1993) CRC Handbook of Microbiological Media (Parks ed.) CRC press, BocaRaton, Fl. Mammalian cell systems often will be in the form ofmonolayers of cells, although mammalian cell suspensions are also used.Alternatively, cells can be derived from those stored in a cell bank(e.g., a blood bank). Illustrative examples of mammalian cell linesinclude the HEC-1-B cell line, VERO and Hela cells, Chinese hamsterovary (CHO) cell lines, W138, BHK, Cos-7 or MDCK cell lines (see, e.g.,Freshney, supra).

In one particularly preferred embodiment, stem cells are used in ex-vivoprocedures for cell transformation and gene therapy. The advantage tousing stem cells is that they can be differentiated into other celltypes in vitro, or can be introduced into a mammal (such as the donor ofthe cells) where they will engraft in the bone marrow. Methods fordifferentiating stem cells (e.g., CD34+) stem cells in vitro intoclinically important immune cell types using cytokines such a GM-CSF,IFN-gamma and TNF-alpha are known (see, e.g., Inaba (1992) J. Exp. Med.,176:1693-1702; Szabolcs (1995) 154:5851-5861).

Rather than using stem cells, T cells or B cells are also used in someembodiments in ex vivo procedures. Several techniques are known forisolating T and B cells. The expression of surface markers facilitatesidentification and purification of such cells. Methods of identificationand isolation of cells include FACS, incubation in flasks with fixedantibodies which bind the particular cell type and panning with magneticbeads.

Stem cells are isolated for transduction and differentiation using knownmethods. For example, in mice, bone marrow cells are isolated bysacrificing the mouse and cutting the leg bones with a pair of scissors.Stem cells are isolated from bone marrow cells by panning the bonemarrow cells with antibodies which bind unwanted cells, such as CD4⁺ andCD8⁺ (T cells), CD45⁺ (panb cells), GR-1 (granulocytes), and Ia^(d)(differentiated antigen presenting cells). For an example of thisprotocol see, e.g., Inaba (1992) supra.

In humans, bone marrow aspirations from iliac crests are performed,e.g., under general anesthesia in the operating room. The bone marrowaspirations is approximately 1,000 ml in quantity and is collected fromthe posterior iliac bones and crests. If the total number of cellscollected is less than about 2×10⁸/kg, a second aspiration using thesternum and anterior iliac crests in addition to posterior crests isperformed. During the operation, irradiated packed red cells areadministered to replace the volume of marrow taken by the aspiration.Human hematopoietic progenitor and stem cells are characterized by thepresence of a CD34 surface membrane antigen. This antigen is used forpurification, e.g., on affinity columns which bind CD34. After the bonemarrow is harvested, the mononuclear cells are separated from the othercomponents by means of ficol gradient centrifugation. This can beperformed by a semi-automated method using a cell separator (e.g., aBaxter Fenwal CS3000+ or Terumo machine). The light density cells,composed mostly of mononuclear cells are collected and the cells areincubated in plastic flasks at about 37° C. for about 1.5 hours. Theadherent cells (monocytes, macrophages and B-Cells) are discarded. Thenon-adherent cells are then collected and incubated with a monoclonalanti-CD34 antibody (e.g., the murine antibody 9C5) at 4° C. for 30minutes with gentle rotation. The final concentration for the anti-CD34antibody is preferably about 10 μg/ml. After two washes, paramagneticmicrospheres (e.g., Dyna Beads, supplied by Baxter Immunotherapy Group,Santa Ana, Calif.) coated with sheep antimouse IgG (Fc) antibody areadded to the cell suspension at a ratio of about 2 cells/bead. After afurther incubation period of about 30 minutes at about 4° C., therosetted cells with magnetic beads are collected with a magnet.Chymopapain (Baxter Immunotherapy Group, Santa Ana, Calif.) at a finalconcentration of 200 U/ml can be added to release the beads from theCD34⁺ cells.

Alternatively, and preferably, an affinity column isolation procedurecan be used which binds to CD34, or to antibodies bound to CD34 (see,e.g., Ho (1995) Stem Cells, 13 (suppl. 3): 100-105, and Brenner (1993)Journal of Hematotherapy, 2: 7-17).

In another embodiment, hematopoietic stem cells can be isolated fromfetal cord blood. Yu (1995) Proc. Natl. Acad. Sci. USA, 92: 699-703describe a preferred method of transducing CD34⁺ cells from human fetalcord blood using retroviral vectors.

3. Administration of Tumor Suppressor-Expressing Nucleic Acid: Vectorsand Expression Cassettes

Routes of Administration

Expression cassettes and vectors (e.g., retroviruses, adenoviruses,liposomes, etc.) containing the therapeutic, tumor suppressor-expressingnucleic acids of the invention, can be administered directly to theorganism for transduction of cells in vivo. Administration is by any ofthe routes normally used for introducing a molecule into ultimatecontact with blood or tissue cells, e.g., systemically, regionally, orlocally, as discussed in detail, supra, for the administration ofadjunctive anti-cancer agents. The “packaged” nucleic acids (at aminimum, a tumor suppressor coding sequence with a promoter) areadministered in any suitable manner, preferably with pharmaceuticallyacceptable carriers, also discussed supra. Suitable methods ofadministering such packaged nucleic acids are available and well knownto those of skill in the art, and, although more than one route can beused to administer a particular composition, a particular route canoften provide a more immediate and more effective reaction than anotherroute.

For example, administration of a recombinant adenovirus vectorengineered to express a tumor suppressor gene can elicit an immuneresponse, specifically, an antibody response, against the adenoviralvector. Some patients may have pre-existing anti-adenoviral reactingantibodies. Thus, in some circumstances, regional or local, rather thansystemic, administration of the tumor-suppressor expressing adenoviralvector is optimal and most effective. For example, as discussed below,ovarian cancer limited to the abdominal cavity is one clinical scenarioin which regional p53 gene therapy, i.e., intraperitoneal (IP)administration, should be considered as a preferred treatment plan.Administration of recombinant adenoviruses IP also results in infectionof the peritoneal lining and absorption of the adenoviral vector intothe systemic circulation (other means of regional administration canalso result in introduction of the adenoviral vector into the systemiccirculation). The extent of this effect may depend on the concentrationand/or total amount of viral particles administered IP. If the systemiceffect is desired, a higher concentration over several consecutive daysmay be preferred.

Local administration of the tumor suppressor-expressing adenoviralvector of the invention is also preferred in some circumstances, e.g.,when the patient has pre-existing anti-adenoviral reactive antibodies.Such “local administration” can be, e.g., by intra-tumoral injection, ifinternal, or mucosal application, if external. Alternatively, a “localadministration” effect can be effected by targeting the adenoviralvector to the tumor using, e.g., tumor specific antigen-recognizingreagents (as antibodies) on liposomes or on the adenovirus itself.

Formulations

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

Formulations suitable for oral administration of pharmaceuticalcompositions comprising the tumor suppressor-expressing nucleic acidscan consist of (a) liquid solutions, such as an effective amount of thepackaged nucleic acid suspended in diluents, such as water, saline orPEG 400; (b) capsules, sachets or tablets, each containing apredetermined amount of the active ingredient, as liquids, solids,granules or gelatin; (c) suspensions in an appropriate liquid; and (d)suitable emulsions. Tablet forms can include one or more of lactose,sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potatostarch, tragacanth, microcrystalline cellulose, acacia, gelatin,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, stearic acid, and other excipients, colorants, fillers,binders, diluents, buffering agents, moistening agents, preservatives,flavoring agents, dyes, disintegrating agents, and pharmaceuticallycompatible carriers. Lozenge forms can comprise the active ingredient ina flavor, usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin or sucrose and acacia emulsions, gels, and the like containing,in addition to the active ingredient, carriers known in the art.

The packaged nucleic acids, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for rectal administration include, for example,suppositories, which consist of the packaged nucleic acid with asuppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. In addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the packaged nucleic acid with a base, including, forexample, liquid triglycerides, polyethylene glycols, and paraffinhydrocarbons.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials.

Formulations of the invention as injection solutions and suspensions canbe prepared from sterile powders, granules, and tablets of the kindpreviously described. The exact composition of the formulation, theconcentration of the reagents and nucleic acid in the formulation, itspH, buffers, and other parameters will vary depending on the mode andsite of administration (e.g., whether systemic, regional or localadministration) and needs related to storage, handling, shipping, andshelf life of the particular pharmaceutical composition. Optimization ofthese parameters depending on the particular need of the formulation canbe done by routine methods; and any of ingredients and parameters forknown injectable formulations can be used. One example of a suitableformulation is, e.g., a recombinant wild-type p53-expressing adenovirusvector of the invention (rAd5/p53) at a concentration of about 7.5×10¹¹to 7.5×10¹⁰ particles per ml, sodium phosphate monohydrate at 0.42mg/ml, sodium phosphate dibasic anhydride at 2.48 mg/ml, sodium chlorideat sodium phosphate monohydrate at 5.8 mg/ml, sucrose at 20.0 mg/ml,magnesium chloride hexahydrate at 0.40 mg/ml, typically stored in 1.0 mldosages. An exemplary formulation for enhanced stability during storageand distribution, especially at refrigeration temperatures, usesrAd5/p53 (at also about 7.5×10¹¹ to 7.5×10¹⁰ particles per ml), sodiumphosphate monobasic dihydrate at 1.7 mg/ml, tromethamine (Trizma, or,Tris base, Sigma Chemical Co., St. Louis, Mo.) at 1.7 mg/ml, magnesiumchloride hexahydrate at 0.4 mg/ml, sucrose at 20 mg/ml, polysorbate 80at 0.15 mg/ml, glycerol at 100 mg/ml, typically stored in 1.0 mldosages.

Cells transduced by the packaged nucleic acid as described above in thecontext of ex vivo therapy can also be administered intravenously orparenterally as described above.

The dose administered to a patient, in the context of the presentinvention, should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

In determining the effective amount of the vector to be administered inthe treatment, the physician evaluates circulating plasma levels of thevector, vector toxicities, progression of the disease, and theproduction of anti-vector antibodies. The typical dose for a nucleicacid is highly dependent on route of administration and gene deliverysystem. Depending on delivery method the dosage can easily range fromabout 1 μg to 100 mg or more. In general, the dose equivalent of a nakednucleic acid from a vector is from about 1 μg to 100 μg for a typical 70kilogram patient, and doses of vectors which include a viral particleare calculated to yield an equivalent amount of therapeutic nucleicacid.

For administration, transduced cells of the present invention can beadministered at a rate determined by the LD₅₀ of the vector, ortransduced cell type, and the side-effects of the vector or cell type atvarious concentrations, as applied to the mass and overall health of thepatient. Administration can be accomplished via single or divided dosesas described below.

In a preferred embodiment, prior to infusion, blood samples are obtainedand saved for analysis. Vital signs and oxygen saturation by pulseoximetry are closely monitored. Blood samples are preferably obtained 5minutes and 1 hour following infusion and saved for subsequent analysis.In ex vivo therapy, leukopheresis, transduction and reinfusion can berepeated are repeated every 2 to 3 months. After the first treatment,infusions can be performed on a outpatient basis at the discretion ofthe clinician. If the reinfusion is given as an outpatient, theparticipant is monitored for at least 4, and preferably 8 hoursfollowing the therapy.

As described above, the adenoviral constructs can be administeredsystemically (e.g., intravenously), regionally (e.g., intraperitoneally)or locally (e.g., intra- or peri-tumoral or intracystic injection, e.g.,to treat bladder cancer). Particularly preferred modes of administrationinclude intra-arterial injection, more preferably intra-hepatic arteryinjection (e.g., for treatment of liver tumors), or, where it is desiredto deliver a composition to a brain tumor, a carotid artery or an arteryof the carotid system of arteries (e.g., occipital artery, auricularartery, temporal artery, cerebral artery, maxillary artery, etc.).Delivery for treatment of lung cancer can be accomplished, e.g., by useof a bronchoscope. Typically such administration is in an aqueouspharmacologically acceptable buffer as described above. However, on oneparticularly preferred embodiment, the adenoviral constructs or thetumor suppressor expression cassettes are administered in a lipidformulation, more particularly either complexed with liposomes to forlipid/nucleic acid complexes (e.g., as described by Debs and Zhu (1993)WO 93/24640; Mannino (1988) supra; Rose, U.S. Pat. No. 5,279,833;Brigham (1991) WO 91/06309; and Felgner (1987) supra) or encapsulated inliposomes, more preferably in immunoliposomes directed to specific tumormarkers. It will be appreciated that such lipid formulations can also beadministered topically, systemically, or delivered via aerosol.

4. Enhancing Tumor Suppressor Delivery

Tumor suppressor delivery can be enhanced by the use of one or more“delivery-enhancing agents”. A “delivery-enhancing agent” refers to anyagent which enhances delivery of a therapeutic gene, such as a tumorsuppressor gene to a cancerous tissue or organ. Such enhanced deliverymay be achieved by various mechanisms. One such mechanism may involvethe disruption of the protective glycosaminoglycan layer on theepithelial surface of an organ or tissue (e.g., the bladder). Examplesof such delivery-enhancing agents are detergents, alcohols, glycols,surfactants, bile salts, heparin antagonists, cyclooxygenase inhibitors,hypertonic salt solutions, and acetates. Alcohols include, for example,the aliphatic alcohols such as ethanol, N-propanol, isopropanol, butylalcohol, acetyl alcohol. Glycols include glycerine, propyleneglycol,polyethyleneglycol and other low molecular weight glycols such asglycerol and thioglycerol. Acetates such as acetic acid, gluconolacetate, and sodium acetate are further examples of delivery-enhancingagents. Hypertonic salt solutions like 1M NaCl are also examples ofdelivery-enhancing agents. Examples of surfactants are sodium dodecylsulfate (SDS) and lysolecithin, polysorbate 80,nonylphenoxypolyoxyethylene, lysophosphatidylcholine, polyethylenglycol400, polysorbate 80, polyoxyethylene ethers, polyglycol ethersurfactants and DMSO. Bile salts such as taurocholate, sodiumtauro-deoxycholate, deoxycholate, chenodesoxycholate, glycocholic acid,glycochenodeoxycholic acid and other astringents like silver nitrate maybe used. Heparin-antagonists like quaternary amines such as prolaminesulfate may also be used. Cyclooxygenase inhibitors such as sodiumsalicylate, salicylic acid, and non-steroidal anti-inflammatory drug(NSAIDS) like indomethacin, naproxen, diclofenac may be used.

Detergents include anionic, cationic, zwitterionic, and nonionicdetergents. Exemplary detergents include but are not limited totaurocholate, deoxycholate, taurodeoxycholate, cetylpyridium,benalkonium chloride, ZWITTERGENT®3-14 detergent, CHAPS(3-[(3-Cholamidopropyl)dimethylammoniol)-1-propanesulfonate hydrate,Aldrich), Big CHAP (as described in U.S. Ser. No. 08/889,355, filed Jul.8, 1997; and, International Application WO 97/25072, Jul. 17, 1997),Deoxy Big CHAP (ibid), TRITON®-X-100 detergent, C12E8,Octyl-B-D-Glucopyranoside, PLURONIC®-F68 detergent, TWEEN® 20 detergent,and TWEEN® 80 detergent (CALBIOCHEM® Biochemicals).

In an embodiment, the delivery-enhancing agent is included in the bufferin which the recombinant adenoviral vector delivery system isformulated. The delivery-enhancing agent may be administered prior tothe recombinant virus or concomitant with the virus. In someembodiments, the delivery-enhancing agent is provided with the virus bymixing a virus preparation with a delivery-enhancing agent formulationjust prior to administration to the patient. In other embodiments, thedelivery-enhancing agent and virus are provided in a single vial to thecare giver for administration.

In the case of a pharmaceutical composition comprising a tumorsuppressor gene contained in a recombinant adenoviral vector deliverysystem formulated in a buffer which further comprises adelivery-enhancing agent, the pharmaceutical composition is preferablybe administered over time in the range of about 5 minutes to 3 hours,preferably about 10 minutes to 120 minutes, and most preferably about 15minutes to 90 minutes. In another embodiment the delivery-enhancingagent may be administered prior to administration of the recombinantadenoviral vector delivery system containing the tumor suppressor gene.The prior administration of the delivery-enhancing agent may be in therange of about 30 seconds to 1 hour, preferably about 1 minute to 10minutes, and most preferably about 1 minute to 5 minutes prior toadministration of the adenoviral vector delivery system containing thetumor suppressor gene.

The concentration of the delivery-enhancing agent will depend on anumber of factors known to one of ordinary skill in the art such as theparticular delivery-enhancing agent being used, the buffer, pH, targettissue or organ and mode of administration. The concentration of thedelivery-enhancing agent will be in the range of 1% to 50% (v/v),preferably 10% to 40% (v/v) and most preferably 15% to 30% (v/v).Preferably, the detergent concentration in the final formulationadministered to the patient is about 0.5-2× the critical micellizationconcentration (CMC). A preferred concentration of Big CHAP is about 2-20mM, more preferable about 3.5-7 mM.

The buffer containing the delivery-enhancing agent may be anypharmaceutical buffer such as phosphate buffered saline or sodiumphosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water andother buffers known to the ordinarily skilled artisan such as thosedescribed by Good et al. (1966) Biochemistry, 5:467. The pH of thebuffer in the pharmaceutical composition comprising the tumor suppressorgene contained in the adenoviral vector delivery system, may be in therange of 6.4 to 8.4, preferably 7 to 7.5, and most preferably 7.2 to7.4.

A preferred formulation for administration of a recombinant adenovirusis about 10⁹-10¹¹ PN/ml virus, about 2-10 mM Big CHAP or about 0.1-1.0mM TRITON®-X-100 detergent, in phosphate buffered saline (PBS), plusabout 2-3% sucrose (w/v) and about 1-3 mM MgCl₂, at about pH 6.4-8.4.The use of delivery-enhancing agents is described in detail in copendingin copending application U.S. Ser. No. 08/779,627 filed on Jan. 7, 1997.

In order to facilitate the improved gene transfer for nucleic acidformulations comprising commercial Big-CHAP preparations; theconcentration of Big CHAP will vary based on its commercial source. Whenthe Big CHAP is sourced from CALBIOCHEM, it is preferred that theconcentration be in a range of 2 to 10 millimolar. More preferred is 4to 8 millimolar. Most preferred is approximately 7 millimolar.

When the Big CHAP is sourced from Sigma, it is preferred that theconcentration of Big CHAP be in a range of 15 to 35 millimolar. Morepreferred is 20 to 30 millimolar. Most preferred is approximately 25millimolar.

In a further embodiment of the invention, delivery-enhancing agentshaving Formula I are provided:

wherein n is an integer from 2-8, X₁ is a cholic acid group ordeoxycholic acid group, and X₂ and X₃ are each independently selectedfrom the group consisting of a cholic acid group, a deoxycholic acidgroup, and a saccharide group. At least one of X₂ and X₃ is a saccharidegroup. The saccharide group may be selected from the group consisting ofpentose monosaccharide groups, hexose monosaccharide groups,pentose-pentose disaccharide groups, hexose-hexose disaccharide groups,pentose-hexose disaccharide groups, and hexose-pentose disaccharidegroups. In one preferred embodiment; the compounds of the presentinvention have the Formula II:

wherein X₁ and X₂ are selected from the group consisting of a cholicacid group and a deoxycholic acid group and X₃ is a saccharide group.

These compounds are preferably used in the range of about 0.002 to 2mg/ml, more preferably about 0.02 to 2 mg/ml, most preferably about 0.2to 2 mg/ml in the formulations of the invention. Most preferred isapproximately 2 mg/ml.

Phosphate buffered saline (PBS) is the preferred solubilizing agent forthese compounds. However, one of ordinary skill in the art willrecognize that certain additional excipients and additives may bedesirable to achieve solubility characteristics of these agents forvarious pharmaceutical formulations. For examples, the addition of wellknown solubilizing agents such as detergents, fatty acid esters,surfactants may be added in appropriate concentrations so as tofacilitate the solubilization of the compounds in the various solventsto be employed. When the solvent is PBS, a preferred solubilizing agentis Tween 80 at a concentration of approximately 0.15%.

5. Administration of Tumor Suppressor Proteins

Tumor suppressor proteins (polypeptides) can be delivered directly tothe tumor site by injection or administered systemically as describedabove. In a preferred embodiment, the tumor suppressor proteins arecombined with a pharmaceutically acceptable carrier (excipient) to forma pharmacological composition as described above. The tumor suppressorpolypeptide will be administered in a therapeutically effective dose.Thus the compositions will be administered in an amount sufficient tocure or at least partially arrest the disease and/or its complications.Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's health.

It will be recognized that tumor suppressor polypeptides, whenadministered orally, must be protected from digestion. This is typicallyaccomplished either by complexing the polypeptide with a composition torender it resistant to acidic and enzymatic hydrolysis or by packagingthe polypeptide in an appropriately resistant carrier such as a liposomeas described above. Means of protecting polypeptides for oral deliveryare well known in the art (see, e.g., U.S. Pat. No. 5,391,377 describinglipid compositions for oral delivery of therapeutic agents).

III. Combination Pharmaceuticals

The tumor suppressor and the adjunctive anti-cancer agent can beadministered individually with either the tumor suppressor nucleic acidor polypeptide being administered before the adjunctive anti-cancer(tumor suppressor pretreatment) or the adjunctive anti-cancer beingadministered before the tumor suppressor nucleic acid and/or polypeptide(cancer drug pretreatment). Of course the tumor suppressor nucleic acidand/or polypeptide and the adjunctive anti-cancer agent can beadministered simultaneously.

In one embodiment, the tumor suppressor nucleic acid and/or polypeptideand the adjunctive anti-cancer agent are administered as a singlepharmacological composition. In this embodiment, the tumor suppressornucleic acid and/or polypeptide and the adjunctive and cancer agent canbe suspended or solubilized in a single homogeneous delivery vehicle.Alternatively, the tumor suppressor nucleic acid and/or polypeptide andthe adjunctive anti-cancer agent can each be suspended or solubilized indifferent delivery vehicles which in turn are suspended (disbursed) insingle excipient either at the time of administration or continuously.Thus, for example, an adjunctive anti-cancer agent may be solubilized ina polar solvent (e.g., paclitaxel in ethanol) and the tumor suppressornucleic acid may be complexed with a lipid which are then either storedtogether in a suspension or, alternatively are combined at the time ofadministration. Various suitable delivery vehicles, excipients, etc.,are described above.

IV. Treatment Regimen: Combined and Individual Therapy

A) Tumor Suppressor Treatment Regimen

It was a discovery of this invention that tumor suppressor nucleic acidsor polypeptides, more particularly tumor suppressor nucleic acids showgreater efficacy in inhibiting tumor growth when administered inmultiple doses rather than in a single dose. Thus this inventionprovides a treatment regimen for a tumor suppressor gene or polypeptidethat comprises multiple administrations of the tumor suppressor nucleicacid or polypeptide.

The tumor suppressor protein or tumor suppressor nucleic acid may beadministered (with or without an adjunctive anti-cancer agent) in atotal dose ranging from about 1×10⁹ to about 1×10¹⁴, about 1×10⁹ toabout 7.5×10¹⁵ preferably about 1×10¹¹ to about 7.5×10¹³, adenovirusparticles in a treatment regimen selected from the group consisting of:the total dose in a single dose, the total dose divided over 5 days oradministered daily for 5 days, the total dose divided over 15 days oradministered daily for 15 days, and the total dose divided over 30 daysor administered daily for 30 days. This method of administration can berepeated for two or more cycles (more preferably for three cycles) andthe two or more cycles are can be spaced apart by three or four weeks.The treatment may consist of a single dosage cycle or dosage cycles mayrange from about 2 to about 12, more preferably from about 2 to about 6cycles.

Particularly preferred treatment regimen include the total dose dividedover 5 days and administered daily, the total dose divided over 15 daysand administered daily, and the total dose divided over 30 days andadministered daily.

In some preferred embodiments, a daily dose in the range of 7.5×10⁹ toabout 7.5×10¹⁵, preferably about 1×10¹² to about 7.5×10¹³, adenovirusparticles can be administered each day for up to 30 days (e.g., aregimen of 2 days, 2 to 5 days, 7 days, 14 days, or 30 days with thesame dose being administered each day). The multiple regimen can berepeated in recurring cycles of 21 to 28 days.

In some embodiments, different routes of administration will result inuse of different preferred dosage ranges. For instance, forintra-hepatic arterial delivery, a preferred range will typically bebetween 7.5×10⁹ and about 1×10¹⁵, more preferably about 1×10¹¹ to about7.5×10¹³, adenovirus particles per day for 5 to 14 days. These regimenscan further include administration of adjunctive anti-cancer agents,FUDR or 5′-deoxy-5-fluorouridine (5′-DFUR), or irinotecan hydrochloride(CPT-11; 7-ethyl-10-[4-(1piperidino)-1-piperidino]carbonyloxycamptothecin). For intratumoraldelivery, a preferred range will typically be between 7.5×10⁹ and about1×10¹³, more preferably about 1×10¹¹ to about 7.5×10¹², adenovirusparticles per day. For intraperitoneal delivery, a preferred range willtypically be between 7.5×10⁹ and about 1×10¹⁵, more preferably about1×10¹¹ to about 7.5×10¹³, adenovirus particles per day for 5-10 days.

B) Combination Therapy Treatment Regimen

Where the tumor suppressor is used in combination with an adjunctiveanti-cancer agent the tumor suppressor nucleic acid is administered intotal dose as described above. In combination, the adjunctiveanti-cancer agent is administered in a total dose dependent upon theagent used. For instance, paclitaxel or a paclitaxel derivative isadministered in a total dose ranging from 75-350 mg/m² over 1 hour, 3hours, 6 hours, or 24 hours in a treatment regimen selected from thegroup consisting of administration in a single dose, in a doseadministered daily on day 1 and day 2, in a dose administered daily onday 1, day 2, and day 3, on a daily dosage for 15 days, on a dailydosage for 30 days, on daily continuous infusion for 15 days, on dailycontinuous infusion for 30 days. A preferred dose is 100-250 mg/m² in 24hours.

Pretreatment with an adjunctive anti-cancer agent (e.g., paclitaxel)prior to treatment with a tumor suppressor nucleic acid enhances theefficacy of the tumor suppressor. Thus, in one particularly preferredembodiment the cell, tissue, or organism is treated with the adjunctiveanti-cancer agent prior to the tumor suppressor nucleic acid. Theadjunctive anti-cancer agent treatment preferably precedes the tumorsuppressor nucleic acid treatment by about twenty four hours althoughlonger or shorter periods are acceptable.

The pretreatment is particularly efficacious when the adjunctiveanti-cancer agent is a paclitaxel-like compound, more preferablypaclitaxel or a paclitaxel derivative (e.g., TAXOL® or TAXOTERE®.Particularly preferred tumor suppressors are RB and p53 with p53 beingmost preferred, in particular p53 in an adenoviral vector (e.g.,A/C/N/53).

V. Treatment of and Prophylaxis for Metastases

As illustrated in Examples 2 and 3, tumor suppressor (e.g., p53) genereplacement therapy has been demonstrated to have efficacy against humantumor cells in vitro, human tumor xenografts in immunocompromised hosts,and human lung tumors (in vivo). Surgical debulking of primary tumors inpatients often results in tumor regrowth at the primary site and tumormetastasis from that site due to microscopic “nests” of tumor cellswhich are missed by the surgeon. Alternatively, in order to make surethat all the tumor is removed from a primary site, the patient may besubjected to disfiguring surgery which removes a large amount of normaltissue surrounding the primary tumor site.

In another embodiment, this invention provides methods of inhibiting thegrowth. and/or proliferation of metastases (metastatic cells). Themethod generally involves either systemic or topical administration of atumor suppressor, more preferably topical administration of p53 or RB.

A) Systemic Treatment.

As explained in Examples 2 and 3, systemic treatment (e.g., intravenousinjection) of tumor suppressor vectors (e.g., A/C/N/53) inhibited theprogression of metastases in vivo. Thus, in one embodiment, thisinvention provides methods for inhibiting the progression of metastaticdisease by administering to an organism a tumor suppressor nucleic acidand/or a tumor suppressor polypeptide as described above. The tumorsuppressor is preferably a tumor suppressor nucleic acid, morepreferably a p53 tumor suppressor nucleic acid and most preferably a p53nucleic acid in an adenoviral vector (e.g. A/C/N/53). In anotherpreferred embodiment, the tumor suppressor nucleic acid is providedencapsulated in a liposome or complexed to a lipid (see, e.g., Debs andZhu (1993). WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques6(7): 682-691; Rose U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309;and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414).

B) Topical Treatment

In another embodiment, topical application of the tumor suppressorprotein or tumor suppressor nucleic acid is preferred in conjunctionwith surgery. In this embodiment, the tumor suppressor, preferably inthe form of an infectious vector, is applied along the surface of thewound cavity after tumor removal. The infectious particles will carryp53 into any residual tumor cells at the wound site, inducing theirapoptosis (programmed cell death). This treatment will impact long-termpatient survival and/or reduce the amount of normal tissue surroundingthe tumor site which needs to be removed during surgery.

The tumor suppressor is preferably compounded in one of the manyformulations known by those of skill in the art to be suitable fortopical application. Thus, for example, an infections preparation of thehuman p53 tumor suppressor gene (e.g., A/C/N/53) is suspended in asuitable vehicle (e.g., petroleum jelly or other cream or ointment)which is suitable for spreading along the surface of the wound cavity.Alternatively, the tumor suppressor can be prepared in an aerosolvehicle for application as a spray inside the wound cavity. In otherembodiments, the tumor suppressor can be prepared in degradable(resorbable) materials, e.g,. resorbable sponges, that can be packedinto the wound cavity and which release the tumor suppressor protein orvector in a time-dependent manner.

Preferred embodiments for application of recombinant adenoviral vectorsto certain defined topical areas, e.g., cornea, gastro-intestinal tract,tumoral resection sites use solid carriers to support a longerincubation time and facilitate viral infection. Carriers can be gauze orointments soaked with the recombinant adenovirus solution. The virus canbe applied via the gauze support to the cornea to achieve improvedtransgene effects. The drained gauze can also be prophylacticallyapplied to resected tumor areas in order to avoid recurrence. Ointmentscan be applied topically to areas of the gastrointestinal tract, ortopically to areas of the pancreas for tumor suppressor gene therapy.

Exemplary ointment carriers include petroleum based PURALUBED or watersoluble KY-JELLY®. In an exemplary method, sterile gauze pads (5×5 cm)or tear flow test strips can be soaked in an adenoviral vector solution(e.g., 1×10⁹ PN/ml) until totally wet. The pads or strips are layered ontop of the target tissue and incubated at 37 degrees C. for 30 minutes.One of skill will recognize that other fabrics, gelatins, or ointmentscan be included that can take up or be mixable with water. In addition,other excipients may be added that can enhance gene transfer asdescribed above.

VI. Combination Treatments with Other Chemotherapeutics

A) Tumor Suppressors Administered in Combination with MultipleChemotherapeutic Combinations

It will be appreciated that the methods of this invention are notlimited to combination of a tumor suppressor with a single adjunctiveanti-cancer agent. While methods typically involve contacting a cellwith a tumor suppressor (e.g., p53) and an adjunctive anti-cancer agentsuch as paclitaxel, the methods of the invention also entail contactingthe cell with a combination of a tumor suppressor gene or polypeptideand two, three or a multiplicity of adjunctive anti-cancer agents andoptionally other chemotherapeutic drugs. In addition, one of skill willrecognize that a chemotherapeutic agent(s) can also be used with tumorsuppressor proteins or genes, in the absence of an adjunctiveanti-cancer agent(s).

Many chemotherapeutic drugs are well known in the scientific and patentliterature; exemplary drugs that can be used in the methods of theinvention include but are not limited to: DNA damaging agents (includingDNA alkylating agents) e.g., cisplatin, carboplatin (see, e.g., Duffull(1997) Clin Pharmacokinet. 33:161-183); Droz (1996) Ann Oncol.7:997-1003), navelbine (vinorelbine), Asaley, AZQ, BCNU, Busulfan,carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil,chlorozotocin, cis-platinum, clomesone, cyanomorpholino-doxorubicin,cyclodisone, cytoxan, dianhydrogalactitol, fluorodopan, hepsulfam,hycanthone, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogenmustard, PCNU, piperazine alkylator, piperazinedione, pipobroman,porfiromycin, spirohydantoin mustard, teroxirone, tetraplatin,thio-tepa, triethylenemelamine, uracil nitrogen mustard, Yoshi-864);topoisomerase I inhibitors (e.g., topotecan hydrochloride, irinotecanhydrochloride (CPT 11), camptothecin, camptothecin Na salt,aminocamptothecin, CPT-11 and other camptothecin derivatives);topoisomerase II inhibitors (doxorubicin, including doxorubicinencapsulated in liposomes (see, U.S. Pat. Nos. 5,013,556 and 5,213,804)amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine,bisantrene HCL, daunorubicin, deoxydoxorubicin, mitoxantrone, menogaril,N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26, and VP-16);RNA/DNA antimetabolites (e.g., L-alanosine, 5-azacytidine,5-fluorouracil, acivicin, aminopterin, aminopterin derivatives, anantifol, Baker's soluble antifol, dichlorallyl lawsone, brequinar,ftorafur (pro-drug), 5,6-dihydro-5-azacytidine, methotrexate,methotrexate derivatives, N-(phosphonoacetyl)-L-aspartate (PALA),pyrazofurin, and trimetrexate); and, DNA antimetabolites (e.g., 3-HP,2′-deoxy-5-fluorouridine, 5-HP, alpha-TGDR, aphidicolin glycinate,ara-C, 5-aza-2′-deoxycytidine, beta-TGDR, cyclocytidine, guanazole,hydroxyurea, inosine, glycodialdehyde, macbecin II, pyrazoloimidazole,thioguanine, and thiopurine). The tumor suppressor nucleic acid and/orpolypeptide can also be administered in combination withchemotherapeutic agents such as vincristine, temozolomide (see, e.g.,U.S. Pat. No. 5,260,291), and toremifene (see, e.g., U.S. Pat. No.4,696,949 for information on toremifene).

Preclinical studies in relevant animal models have. shown that p53adenovirus combined with cisplatin, carboplatin, navelbine, doxorubicin,5-fluorouracil, methotrexate, or etoposide, inhibited cell proliferationmore effectively than chemotherapy alone treating the tumors: SSC-9 headand neck, SSC-15 head and neck, SSC-25 head and neck, SK OV-3 ovarian,DU-145 prostate, MDA-MB-468 breast and MDA-MB-231 breast tumor cells. Inanother embodiment, an enhanced anti-tumor efficacy is seen using athree drug combination of p53 gene (expressed, e.g., in an adenovirasvector), an adjuctive anti-cancer agent (e.g., paclitaxel) and a DNAdamaging agent (e.g., cisplatin). The combination of p53, paclitaxel andcisplatin has been shown to be effective in an ovarian tumor model.These data support the combination of p53 gene therapy with chemotherapyin clinical trials.

These other chemotherapeutic drugs can be used in combination with thetumor suppressor nucleic acid and/or polypeptide with or without thepresence of an adjunctive anti-cancer agent. This invention alsocontemplates the use of radiation therapy in combination with any of thetumor suppressors described above or in conjunction with the tumorsuppressors described above combined with an adjunctive anti-canceragent.

It will also be appreciated that any of these chemotherapeutics can beused individually in combination with a tumor suppressor nucleic acid orpolypeptide according to the methods of this invention.

When the tumor suppressor nucleic acid (e.g., p53) is administered in anadenoviral vector with an adjunctive anti-cancer agent (e.g.,paclitaxel) and a DNA damaging agent (e.g., cisplatin, carboplatin, ornavelbine), the adenoviral vector is typically administered for 5 to 14days at about 7.5×10¹² to about 7.5×10¹³ adenoviral particles per day.For example, a daily dose of about 7.5×10¹³ adenoviral particles incombination with carboplatin can be used. In one embodiment, a dailydose of about 7.5×10¹² adenoviral particles can be used foradministration to the lung. In another embodiment, p53 is administeredwith topotecan.

Typically, the DNA damaging agent will be administered at therecommended dose, see, e.g., Physician's Desk Reference, 51 st ed.(Medical Economics, Montvale, N.J. 1997). For instance, carboplatin isadministered to achieve an AUC (an area under the curve) of about 6-7.5mg/ml/min.

Protease Inhibitors

In still another embodiment, this invention provides for the combineduse of tumor suppressor nucleic acids and/or polypeptides and proteaseinhibitors. Particularly preferred protease inhibitors include, but arenot limited to collagenase inhibitors, matrix metalloproteinase (MMP)inhibitors (see, e.g., Chambers (1997) J. Natl. Cancer Inst.89:1260-1270). In a preferred embodiment, the methods compriseadministering concurrently or sequentially, an effective amount of aprotease inhibitor and an effective amount of a tumor suppressorpolypeptide and/or nucleic acid. Examples of compounds that are proteaseinhibitors are well known in the scientific and patent literature.

Immunomodulators

The tumor suppressor proteins and nucleic acids of this invention can beused in conjunction with immunomodulators where the immunomodulatorseither upregulate an immune response directed against thehyperproliferative or cancer cell (e.g., an immune response directedagainst a tumor specific antigen) or downregulate an immune responsedirected against the tumor suppressor protein, tumor suppressor nucleicacid, tumor suppressor vector (e.g., anti-adenoviral reaction), and/orcombined chemotherapeutic.

Thus, for example, this invention provides for the combined sequentialor concurrent administration of an effective amount of a tumorsuppressor nucleic acid and/or tumor suppressor polypeptide with aneffective amount of an immunomodulator. Immunomodulators include, butare not limited to cytokines such as IL-2, IL-4, IL-10 (U.S. Pat. No.5,231,012; Lalani (1997) Ann. Allergy Asthma Immunol. 79:469-483;Geissler (1996) Curr. Opin. Hematol. 3:203-208), IL-12 (see, e.g.,Branson (1996) Human Gene Ther. 1:1995-2002), and gamma-interferon.

Immunomodulators that function as immunosuppressants can be utilized tomitigate an immune response targeted against the therapeutic (e.g.,tumor suppressor protein or nucleic acid or adjunctive anticancer agent,etc.). Immunosuppressants are well known to those of skill in the art.Suitable immunosuppressaats include, but are not limited tocyclo-phosphamide, dexamethasone, cyclosporin, FK506 (tacrolimus)(Lochmuller (1996) Gene Therapy 3:706-716) IL-10, and the like.Antibodies against cell surface receptors which modulate the immuneresponse can also be used. For instance, antibodies that block ligandbinding to cellular receptors on B cells, T cells, NK cells,macrophages, and tumor cells can be used for this purpose. For examplesof this strategy see, e.g., Yang (1996) Gene Therapy 3:412-420; Lei(1996) Human Gene Therapy 7:2273-2279; Yang (1996) Science275:1862-1867.

VII. Therapeutic Kits.

In another embodiment, this invention provides for therapeutic kits. Thekits include, but are not limited to a tumor suppressor nucleic acid orpolypeptide or a pharmaceutical composition thereof. The kits may alsoinclude an adjunctive anti-cancer agent or a pharmaceutical compositionthereof or pharmaceutical composition thereof. The various compositionsmay be provided in separate containers for individual administration orfor combination before administration. Alternatively the variouscompositions may be provided in a single container. The kits may alsoinclude various devices, buffers, assay reagents and the like forpractice of the methods of this invention. In addition, the kits maycontain instructional materials teaching the use of the kit in thevarious methods of this invention (e.g., in the treatment of tumors, inthe prophylaxis and/or treatment of metastases, and the like).

The kit can optionally include one or more immunomodulators (e.g.,immunosuppressants). Particularly preferred immunomodulators include anyof the immunomodulators described herein.

VIII. Cells Containing Heterologous Tumor Suppressor Nucleic Acids orPolypeptides, and Other Agents

Further provided by this invention is a transfected or otherwise treatedprokaryotic or eukaryotic host cell, for example an animal cell (e.g., amammalian cell) containing a heterologous tumor suppressor nucleic acidand/or tumor suppressor polypeptide. The cell may optionallyadditionally contain an adjunctive anti-cancer agent, e.g. paclitaxel orother microtubule affecting agent.

Suitable prokaryotic cells include, but are not limited to bacterialcells such as E. coli cells. Suitable animal cells preferably includemammalian, more preferably human cells. Host cells include, but are notlimited to any mammalian cell, more preferably any neoplastic or tumorcell such as any of the cells described herein.

The transfected host cells described herein are useful as compositionsfor diagnosis or therapy. When used pharmaceutically, they can becombined with various pharmaceutically acceptable carriers as describedabove for ex vivo gene therapy. The cells can be administeredtherapeutically or prophylactically in effective amounts described indetail above. In a diagnostic context, the cells may be used forteaching or other reference purposes and provide suitable models foridentification of cells thus transfected and/or treated.

IX. Preclinical and Clinical Efficacy of p53 Adenovirus Gene Therapy

Adenovirus-mediated p53 gene therapy is currently undergoing phase I/IIclinical trials in several countries. The pharmaceutical compositionused in these clinical trials included an exemplary wild typep53-expressing-adenovirus of the invention (rAd/p53) consisting of areplication-deficient, type 5 adenovirus vector expressing the humantumor suppressor gene under the control of the cytomegalovirus promoter(“rAd5/p53”), as described herein (see Wills (1994) supra).

Regional Administration

Ovarian cancer limited to the abdominal cavity is one clinical scenarioin which regional p53 gene therapy, i.e., intraperitonealadministration, should be considered as a preferred treatment plan.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Combination Therapy with p53 and TAXOL®

The invention provides for the combined administration of nucleic acidexpressing a tumor suppressor polypeptide and paclitaxel in thetreatment of neoplasms. The following example details the ability of ap53 expressing adenovirus of the invention in combination with TAXOL® totreat neoplasms, and that the combination therapy was more effective atkilling tumor cells than either agent alone.

Combination Therapy In Vitro.

The cells were subjected to one of three treatment regimes: In treatment1, the cells were pretreated with TAXOL® twenty-four hours beforeexposure to the p53 adenovirus construct A/C/N/53. In treatment two, thecells were pretreated with the p53 adenovirus construct and then latercontacted with TAXOL®. In treatment three, the cells were contactedsimultaneously with both the TAXOL® and the p53 adenovirus. Thus, thep53 Ad and TAXOL® can be administered within the same twenty four (24)hour period or concurrently.

Approximately 1.5×10⁴ cells in culture medium (head and neck cell linesSCC-9, SCC-15, and SCC-25 in 1:1 mix of DMEM+Ham's F12 media with 0.4μg/ml cortisol and 10% FBS and 1% non-essential amino acids, prostateDU-145 and Ovarian SK-OV-3 in Eagles essential medium plus 10% FBS) wereadded to each well on a 96 well microtitre plate and cultured for about4 hours at 37° C. and 5% CO₂. The drug (TAXOL®), the p53 adenovirus, orthe appropriate vehicle/buffer was added to each well. As paclitaxel isnot water soluble, the drug was dissolved in ethyl alcohol prior toadministration. Cells were then cultured overnight at 37° C. and 5% CO₂.p53 adenoviruses were administered in phosphate buffer (20 mM NaH₂PO₄,pH 8.0, 130 mM NaCl, 2 mM MgCl₂, 2% sucrose).

Cell death was then quantitated according to the method of Mosmann(1983) J. Immunol. Meth., 65: 55-63. Briefly, approximately 25 μl of 5mg/ml MTT vital dye [3-(4,5 dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide] was added to each well and allowed to incubate for 3-4 hrs. at37° C. and 5% CO₂. Then 100 μl of 10% SDS detergent was added to eachwell and allowed to incubate overnight at 37° C. and 5% CO₂. Signal ineach well was then quantitated using a Molecular devices microtiterplate reader (Thermo-Max). The particular cell lines used and theresults obtained therefrom are listed in Table 1. TABLE 1 In vitroevaluation of the adjunctive anti-cancer agent TAXOL ® combined withtumor suppressor nucleic acid. TAXOL ® A/C/N/5 Treatment Cell line dose3 dose TAXOL ® Cancer (μg/ml) (m.o.i.) pretreatment p53 pretreatmentsimultaneous SK-OV-3 0.37 40 additive effect no effect additive effectOvarian p ≦ 0.0001 p > 0.2000 p ≦ 0.0001 cancer SCC-25 0.10 or 2.5 oradditive effect very small effect additive effect Head and 0.01 5.0 p ≦0.0001 p = 0.0606 p ≦ 0.0001 neck cancer SCC-15 0.10 or 2.5 or additiveeffect additive effect additive effect Head and 0.01 5.0 p ≦ 0.002 p ≦0.0001 p ≦ 0.0001 neck cancer DU-145 0.36 or 2.5 or additive effectadditive effect additive effect Prostate 0.036 or 5.0 p ≦ 0.03 p ≦0.0001 p ≦ 0.0001 cancer 0.0036 SCC-9 0.12 or 2.5 or additive effectadditive effect additive effect Head and 0.012 or 5.0 p ≦ 0.01 p ≦0.0001 p ≦ 0.0001 neck cancer 0.0012

In general, the p53 adenovirus was more effective when added after orconcurrently with TAXOL® than when it was added first. These resultssuggest a synergistic interaction between A/C/N/53 and TAXOL®.

Isobologram Analysis Establishes Synergistic Effect.

SK-OV-3 (p53 null) ovarian tumor cells were treated with combinations ofTAXOL® and p53/adenovirus (A/C/N/53) as illustrated in Table 2. Dosingwas performed as described above. Cell death was quantitated on day 3using the MTT assay as described above. In addition, a dose responsecurve for p53 Ad alone (using the doses listed in Table 2) was generated(after 2 day cell exposure to the drug) and a dose response curve forTAXOL® alone was performed using the doses listed above (3 day cellexposure to the drug). TABLE 2 Treatment groups for combined TAXOL ® andp53 Ad (A/C/N/53) treatment TAXOL ® p53 AD Group (μg/ml) (m.o.i.) 10.001 0.5 2 0.01 0.5 3 0.1 0.5 4 0.5 0.5 5 1 0.5 6 5 0.5 7 10 0.5 8 200.5 9 0.001 1 10 0.01 1 11 0.1 1 12 0.5 1 13 1 1 14 5 1 15 10 1 16 20 117 0.001 5 18 0.01 5 19 0.1 5 20 0.5 5 21 1 5 22 5 5 23 10 5 24 20 5 250.001 10 26 0.01 10 27 0.1 10 28 0.5 10 29 1 10 30 5 10 31 10 10 32 2010 33 0.001 25 34 0.01 25 35 0.1 25 36 0.5 25 37 1 25 38 5 25 39 10 2540 20 25 41 0.001 50 42 0.01 50 43 0.1 50 44 0.5 50 45 1 50 46 5 50 4710 50 48 20 50

FIG. 1 illustrates the inhibition of cell proliferation (as compared tothe buffer control) as a function of treatment. In general increasingdoses of either TAXOL® or p53 decreased the rate of cell proliferationwith the combination of p53 and TAXOL® having a greater effect thaneither drug alone.

FIG. 2 illustrates an isobologram analysis of these data using theIsobole method as reviewed by Berenbaum (1989) Pharmacol. Rev. 93-141.Synergism between TAXOL® and p53 (A/C/N/53) was observed when cells werepretreated with TAXOL® 24 hours before p53 (A/C/N/53) treatment. In FIG.2, the straight line (isobole for ED₃₀) represents the effects on cellproliferation which would be expected if treatment with the two drugswere merely additive. In fact, the observed effects fall to the lowerleft of the isobole line indicating that lower than predictedconcentrations of each drug were needed and a synergistic interactionbetween the two drugs has occurred.

Example 2 p53 Adenovirus-Mediated Gene Therapy Against Metastases

The invention provides for the administration of nucleic acid expressinga tumor suppressor polypeptide in the treatment of metastases. Thefollowing example details the ability of a p53 expressing adenovirus ofthe invention to infect various tissues in the body and to treatmetastases.

Female scid mice (mice homozygous for the SCID mutation lack both T andB cells due to a defect in V(D)J recombination) were injected with 5×10⁶MDA-MB-231 mammary carcinoma cells into their mammary fat pads. Afterthe primary tumors were well established and had time to metastasize tothe lungs, the primary tumors were surgically removed (on day 11). Micewere treated with intravenous A/C/N/53 or with control buffer on days23, 30, 37, 44 (1 qW) with control buffer or with A/C/N/53 (a p53 in anadenovirus) at 4×10⁸ C.I.U./injection. On day 49, the lungs wereharvested, fixed, stained and examined microscopically.

The results are illustrated below in Table 3. TABLE 3 Inhibition ofMDA-MB-231 lung metastases using AIC/N/53 Treatment No Metastases ≦6Metastases ≧84 Metastases Buffer 11 (65%) 1 (6%)  5 (29%) n = 17A/C/N/53  5 (50%) 4 (40%) 1 (10%) n = 10

The A/C/N/53 treatment decreased the number of metastases in the micethat had them.

In a second experiment, 231 tumors in the mammary fat pads of scid orscid-beige mice were given peritumoral injections with A/C/N/53. A totaldose of 2-4×10⁹ C.I.U. given in 10 injections decreased the number ofmice with lung metastases by 80% in scid mice and 60% in scid-beigemice. Also the number of metastases per mouse was dramatically reducedin mice with any lung tumors at all. As indicated above, intravenousdosing with A/C/N/53 also demonstrated efficacy against lung metastasesin scid mice. These data indicate that cancer gene therapy with A/C/N/53may impact the severity of metastatic disease in addition to decreasingprimary tumor burden.

In another experiment, female scid mice were injected with 5×10⁶MDA-MB-231 mammary tumor cells/mouse into the mammary fat pad on day 0.The primary (mammary) tumors were surgically removed on day 18. The micewere treated with intravenous injections of buffer, beta-gal AD, or p53Ad (A/C/N/53) on days 21, 24, 32, 39, and 36. The virus dose perinjection was 4×10⁸ C.I.U. (A/C/N/53) (PN/C.I.U.=23.3) and 9.3×10⁹particles beta-gal Ad (PN/C.I.U.=55.6; 1.7×10⁸ C.I.U.).

Lungs and livers were harvested on day 51 and fixed in formalin. Tissuesections were evaluated for lung tumors and for liver damage. Majororgans from 2 buffer and 2 beta-gal Ad mice were flash-frozen forcryosectioning and analysis of B-galactosidase enzyme activity. TABLE 4Inhibition of MDA-MB-231 lung metastases using A/C/N/53 Lung Metastasesper Mouse Buffer Gp.* Beta-gal Ad Gp.* p53 Ad Grp.  ≦20 11% (1)  8% (1) 21% (3)  >20 and ≦100 11% (1) 33% (4)  79% (11) >100 and ≦200 33% (3)33% (4)  0% (0) >200 and ≦300 33% (3) 17% (2)  0% (0) >300 11% (1)  8%(1)  0% (0) Total number  9 12  14 evaluated Regrowth of 82% (9/11) 88%(14/16) 100% (14/14) primary tumor*Number of metastases is under-estimated. Multiple tumors had growntogether in these lungs.

The number of metastases per lung in the buffer and beta-gal Ad groupswas not significantly different (p=0.268, see Table 4).

p53 Ad treatment significantly reduced the number of metastases per lungwhen compared to either the buffer of beta-gal Ad groups (p<0.001 andp<0.002, respectively). In addition to the reduction in the number ofmetastases, there was also a dramatic reduction in the size of lungmetastases in the p53 Ad group. In the control groups, tissue sectionsfrom most lungs were >50% occupied by neoplastic tissue and individualtumors were no longer recognizable over large areas of the lungs. Incontrast, lung metastases in most of the p53 Ad group were small andeasily distinguishable as individual tumors.

Adenovirus Tissue Distribution

Liver tissues had the highest number of infected cells (about 50%) andbeta-galactosidase activity was intense. Lung had scattered patches ofinfected cells evenly distributed throughout the tissue. Intestines andstomach had periodic infection of cells in the outer smooth muscle wallsurrounding the organs. There was also beta-galactosidase activity inscattered microvilli along the lumen. The smooth outer muscle wallsurrounding the uterus had periodic infection of cells similar to thatseen in the intestines. Most stromal cells in the ovary were infected.The spleen had scattered beta-galactosidase activity in the smoothmuscle components of the organ. There were very few infected cells (<1%)inside the main bulk of striated heart muscle. There were almost noinfected cells in primary tumors in the mammary fat pad, nor in theunderlying striated muscle. There were no infected cells in the kidney.

Liver Pathology.

All livers were grossly normal when necropsied. There was no overtnecrosis in any liver. However, mice treated with adenovirus did havehepatocellular abnormalities (not present in the buffer group) whichincluded elevated numbers of cells in mitosis, cellular inclusions, andchanges in hepatocyte size and shape.

Example 3 p53 Adenovirus-Mediated Gene Therapy Against Human BreastCancer Xenografts

The invention provides for the treatment of various cancers by theadministration of nucleic acid expressing a tumor suppressorpolypeptide. The following example details the ability of a p53expressing adenovirus of the invention to treat human breast cancer.

Introduction of wild-type p53 into tumors with null or mutant p53 offersa novel strategy for controlling tumor growth. Casey (1991) Oncogene 6:1791-1797, introduced wild-type p53 into breast cancer cells in vitrovia a plasmid DNA vector. The number of MDA-MB-468 (p53^(mut) and T47D(p53^(mut)) colonies arising after plasmid transfection was reduced 50%by wild-type p53. Also, none of the resultant colonies expressed thewild-type p53 transfectant. By contrast, the number of MCF-7 (p53 wt)colonies was not affected. Negrini (1994) Cancer Res. 54: 1818-1824,conducted a similar study using MDA-MB-231 cells. Colony formation wasreduced 50% by transfection with a plasmid containing wild-type p53 andnone of resultant colonies expressed wild-type p53. Paradoxically, inthis study similar results were observed with MCF-7 cells.

In the study described in this example the efficacy of areplication-deficient, recombinant, E1 region-deleted, p53 adenovirus(p53 Ad; (A/C/N/53) Wills (1994) supra) was tested against three humanbreast cancer cell lines expressing mutant p53, MDA-MB-231, MDA-MB-468,and MDA-MB-435. The MDA-MB-231 cells carry an Arg-to-Lys mutation incodon 280 of the p53 gene (Bartek (1990) Oncogene 5: 893-899). TheMDA-MB-468 cells carry an Arg-to-His mutation in codon 273 (Id.). TheMDA-MB-435 cells carry a Gly-to-Glu mutation in codon 266 of the p53gene (Lesoon-Wood (1995) Hum. Gene Ther. 6:395-405).

Previous studies have shown high levels of wild-type p53 expression intumor cells from human breast, ovary, lung, colorectum, liver, brain,and bladder after infection with p53 Ad in vitro (Wills (1994) supra.,Harris et al. (1996) Cancer Gene Therapy 3: 121-130).Adenovirus-mediated p53 expression ultimately resulted in changes incell morphology and the induction of apoptosis in p53 null or mutant p53tumor cells. Infection of 468 breast cancer cells by p53 Ad at 10 m.o.i.(multiplicity of infection) caused almost 100% inhibition of DNAsynthesis by 72 hrs. after infection. In addition, infection with p53 Adin vitro inhibited proliferation of MDA-MB-468 and MDA-MB-231 cells withED₅₀ values of 3±2 and 12±10 m.o.i., respectively. Proliferation ofthree other p53-mutant breast carcinoma lines was also inhibited at lowconcentrations of p53 Ad. The ED₅₀ values were 16±4 m.o.i. for SK-BR-3cells, 3±3 m.o.i. for T-47D cells, and 2±2 m.o.i. for BT-549 cells.Infection of MDA-MB-468 and MDA-MB-231 cells with 30 m.o.i. of anequivalent, recombinant adenovirus expressing E. coli beta-galactosidase(beta-gal), instead of p53, resulted in >67% beta-gal positiveMDA-MB-468 cells and 34-66% beta-gal positive MDA-MB-231 cells. Bycorrelating the percentage of beta-gal positive cells with the p53anti-proliferative effects in a large panel of tumor cells with alteredp53, Harris et al. (supra.) showed a strong positive correlation betweenthe degree of p53-induced inhibition and the degree of adenovirustransduction. In contrast, cell lines expressing normal levels ofwild-type p53 were minimally affected by p53 transduction, independentof the adenovirus transduction rate.

Proliferation of MCF7 and HBL-100 cells, two human mammary cell linescontaining wild-type p53, was relatively unaffected by p53 Adconcentrations greater than or equal to 99 m.o.i. in vitro. In otherwords, growth inhibition of MCF-7 and HBL-100 cells required p53 Adconcentrations at least 8- and 33-fold higher than the ED₅₀ values for−231 and −468 cells, respectively. Using a similar recombinant p53 Ad,Katayose (1995) Clin. Cancer Res. 1:889-897, demonstrated increased p53protein expression, decreased cell proliferation, and increasedapoptotic cell death in −231 cells transduced in vitro. This studyextends these in vitro results with −468 and −231 cells to breast cancerxenografts in vivo. The efficacy of adenovirus-mediated p53 gene therapyis evaluated in another breast cancer cell line (MDA-MB-435) which isresistant to adenovirus transduction in vitro.

Materials and Methods

Cell Lines and Adenovirus Infections In Vitro

The human breast cancer cell lines MDA-MB-231, -468, and -435 wereobtained from ATCC (Rockville, Md., USA). The −231 cells were culturedin DMEM (Life Technologies, Grand Island, N.Y.) with 10% fetal calfserum (FCS; Hyclone, Logan, Utah) at 37° C. and 5% CO₂. The −468 cellswere cultured in Leibovitz's L-15 medium (Life Technologies) containing10% FCS at 37° C. The −435 cells were cultured in Leibovitz's L-15medium with 15% FCS and 10 μg/ml bovine insulin (Sigma Chem. Co., St.Louis, Mo.) at 37° C.

Construction and propagation of the human wild-type p53 expressing andE. coli beta-galactosidase (beta-gal) expressing recombinantadenoviruses (rAd), where transgene expression is directed by the humancytomegalovirus promoter, have been described previously (Wills (1994)supra). Adenoviruses were administered in phosphate buffer (20 mMNaH₂PO₄, pH 8.0, 130 mM NaCl, 2 mM MgCl₂, 2% sucrose). C.I.U. is definedas cellular infectious units. The concentration of infectious viralparticles was determined by measuring viral hexon protein positive 293cells after a 48 hr. infection period (Huyghe (1995) supra).

For in vitro infection studies with p53 Ad, cells were plated at adensity of 1-5×10⁴ cells/well in 12-well tissue culture dishes (BectonDickinson, Lincoln Park, N.J., USA). Cells were transduced with 0, 10,or 50 m.o.i. (multiplicity of infection ═C.I.U./cell) p53 Ad andcultured for 72 hrs. as previously described (Wills (1994) supra.). Forin vitro infection studies with beta-gal Ad, cells were plated at adensity of 1×10⁵ cells/well. The cells were transduced with 0, 10, 50,or 100 m.o.i. beta-gal Ad. After 48 hours, the cells were fixed with0.2% glutaraldehyde (Sigma Chemical Co.) then washed 3 times with PBS(Life Technologies). The cells were then assayed in 1 ml of X-Galsolution [1.3 mM MgCl₂, 15 mM NaCl, 44 mM Hepes buffer, pH 7.4, 3 mMpotassium ferricyanide, and 1 mg/ml X-Gal in N,N-dimethylformamide (10%final conc.)]. X-Gal was purchased from Boehringer Mannheim Corp.,Indianapolis, Ind. All other chemicals were purchased from Sigma.

To determine the percentage of transduced cells, 5 microscope fieldswere counted from each culture well and the average percent expressingbeta-galactosidase was calculated for 3 wells at each m.o.i.

Adenovirus Treatment In Vivo

Athymic female nude mice were purchased from Charles River Laboratories(Wilmington, Mass., USA). All mice were maintained in a VAF-barrierfacility and all animal procedures were performed in accordance with therules set forth in the N.I.H. Guide for the Care and Use of LaboratoryAnimals. Tumor cells were injected subcutaneously or into the mammaryfat pad.

Cell inoculations were: 5×10⁶-231 cells/mouse, 1×10⁷ MDA-MB-468cells/mouse, or 1×10⁷ MDA-MB-435 cells/mouse. Tumors were allowed togrow in vivo for 10-11 days before the start of dosing, except for one−468 experiment where the tumors grew for 33 days before treatmentstarted. Tumor volume was calculated as the product of measurements inthree dimensions. Tumor volumes for different treatment groups on eachday were compared by Student's t test using Statview II software (AbacusConcepts, Berkeley, Calif.). Average percent inhibitions for groupsdosed on days 0-4 and 7-11 were calculated using significant values(p<0.05) from day 14 to the end of the study.

The specific effects of p53 were distinguished from adenovirus vectoreffects by subtracting the average tumor growth inhibition caused bybeta-gal Ad from growth inhibition caused by p53 Ad. All virusinjections were peri/intra-tumoral. In general, two 5-day courses oftumor therapy (i.e., 5 injections) were given to each mouse, separatedby a 2 day “resting period”. In some cases, this dosing regime wasextended for more than 2 weeks and/or buffer vehicle was substituted forvirus for some injections. Tumor growth curves show mean tumorvolume±s.e.m.

Histology and ApopTag® Immunohistochemistry

Tissue samples were fixed in 10% buffered formalin and processedovernight in a Miles VIP Tissue Processor, then imbedded in paraffin.Five micron tissue sections were cut with a Leitz microtome. The slideswere stained with a routine Harris hematoxylin and eosin stain (Luna etal. (1968), Manual of Histologic Staining Methods of the Armed ForcesInstitute of Pathology. New York: McGraw Hill Book Co.).

ApopTag® in situ apoptosis detection kits were purchased from Oncor(Gaithersburg, Md., USA). Samples were assayed as per kit directions.Briefly, deparaffinized, rehydrated tissue sections were treated withOncor Protein Digesting Enzyme, incubated with TdT, and developed usingan avidin-peroxidase kit (rabbit IgG-Sigma Chem. Co. #EXTRA-3) and DAB(Vector Lab. #SK4100). Slides were counterstained with methyl green.

Beta-Galactosidase Assay

Tumors were embedded in TBS (Triangle Biomedical Sciences, Durham, N.C.,USA) and flash frozen in a 2-methylbutane/dry ice bath. Frozen tissuesections (8 μm thick) were fixed in 0.5% glutaraldehyde at 4° C. for 5min. and then assayed for beta-gal expression as described above:

Integrin FACS Analysis

Cells were suspended by treatment with 0.02% EDTA, pelleted, and thenwashed 2× with PBS. Cells were then resuspended at a concentration of1×10⁶ cells/ml and incubated with primary antibodies (final conc.1:250/ml) at 4° C. for 1 hr. Cell suspensions were washed 2× with PBS toremove excess primary antibody. Cells were then incubated withFITC-conjugated rabbit antimouse adjunctive antibody (final conc.1:250/ml, Zymed) at 4° C. for 1 h. Cells were washed as before with PBSand immediately analyzed. Fluorescence was measured with a FACS Vantageflow cytometer (Becton Dickinson, Mountain View, Calif., USA). Sidescatter and forward scatter were determined simultaneously, and all datawere collected with a Hewlett Packard computer equipped with FACSresearch software (Becton Dickenson). Primary antibodies used to detectintegrin receptors were obtained from the following suppliers:anti-alpha_(v), (12084-018, Gibco BRL); anti-beta₃, (550036, BectonDickenson); anti-alpha_(v)beta₃ (MAP1976, Chemicon); anti-beta₁,(550034, Becton Dickenson); and anti-alpha_(v)beta₅ (MAB 1961,Chemicon).

Results

Adenovirus Transduction Efficiency and p53 Growth Inhibition In Vitro

The −231 and −468 cells were both highly transduced in vitro at anm.o.i. of 10. By contrast, −435 cells were rarely transduced, even at100 m.o.i. For −231 cells, 8% (10 m.o.i.), 46% (50 m.o.i.), and 62% (100m.o.i.) of the cells were transduced by beta-gal Ad. For 468 cells, 78%(10 m.o.i.), 84% (50 m.o.i.), and 97% (100 m.o.i.) of the cells weretransduced by beta-gal Ad. For 435 cells, 0.5% (10 m.o.i.), 1% (50m.o.i.), and 1.3% (100 m.o.i.) of the cells were transduced by beta-galAd.

Infection with 50 m.o.i. p53 Ad resulted in nearly complete cell deathin the 231 and 468 cell cultures. By contrast, p53 Ad had no detectableeffect on the growth of 435 cells.

p53 Ad Efficacy Against Human Breast Cancer Xenografts

Adenovirus-mediated p53 gene therapy was highly effective against −231and −468 xenografts (FIGS. 3 a & 3 b). In the 231 experiment, 1 mouse inthe beta-gal Ad group and 3 mice in the p53 Ad group were tumor-free atthe end of the study, and all tumors regressed during p53 Ad treatment.Inhibition of −231 tumor growth averaged 86% (p≦0.01). The component ofgrowth inhibition due to p53 averaged 37%, while adenovirus-specificinhibition averaged 49% (p≦0.01). Inhibition of −468 tumor growthaveraged 74% (p≦0.001). One mouse in the p53 Ad group was tumor-free atthe end of the study and all tumors regressed during p53 Ad treatment.The component of growth inhibition due to p53 averaged 45% (p<0.001),while adenovirus-specific inhibition averaged 28% (p≦0.05). Noside-effects were observed in either experiment. The ED₅₀ values for−231 and −468 tumor growth inhibition were 3×10⁸ C.I.U. (cell infectiousunits) and 2×10⁸ C.I.U., respectively (FIG. 4). The −435 tumors werealmost completely resistant to p53 Ad treatment (FIG. 3 c). Growthinhibition in the 435 tumor groups treated with adenovirus was notsignificant,

FIG. 5 shows a comparison of the efficacy of two p53 Ad dosing regimesagainst −231 tumors. All mice were given 5 peritumoral injections perweek. All mice treated with the therapeutic agent (p53 Ad) received atotal of 2.2×10⁹ C.I.U./mouse per week. One group received a singlebolus injection containing the entire week's dose of adenovirus. Theother 4 injections for the week consisted of buffer vehicle (I× group).The other treated group received the same Ad dose split into fiveinjections per week (5× group). This dosing regime was given duringweeks 1 and 3 (days 0-4, 14-18). Growth inhibition averaged 73% for the5× group (p−<0.01), but only 44% for the 1× group (p<0.05 for the firstthree weeks of the study, not significant after day 21). The first cycleof p53 gene therapy was more effective than the second cycle. After thefirst therapy cycle, 4 mice in the 1× group, 5 mice in the 5× group, and1 mouse in the vehicle control group were tumor-free. One mouse in the5× group relapsed with a very small tumor by day 21. No further “cures”were observed after the second cycle of therapy.

FIG. 6 shows an experiment using 468 tumors that were initially 4-foldlarger than the 468 tumors shown in FIG. 3 b, treated with a 10-foldlower dose of adenovirus. A total dose of 2.2×10⁸ C.I.U. p53 Ad/mouseper week was administered. One group received a single bolus injectionof virus, followed by 4 injections of buffer per week (I× group). Theother treated group received the same viral dose split into fiveinjections per week (5× group). These dosing schedules were given for 6weeks. The total viral dose administered over 6 weeks was approximatelyhalf the dose used in FIG. 3. This dosing regime resulted in acytostatic effect on tumor volume in mice treated with p53 Ad (p≦0.05).Treatments given early in the study appeared to be more effective thanthose given during later weeks. One mouse in the 5× group was tumor-freeby day 21. However when tumor growth inhibition in all mice wascompared, the I× dosing regime (60%) was slightly, but notsignificantly, more effective than the 5× regime (55%). By 1 week afterthe end of dosing, the tumor growth rate in the 5× group started toincrease. One month after the start of the study, the vehicle controltumors started to necrose and growth plateaued.

In Vivo Infectivity After Repeated Adenovirus Exposure

At the end of the studies shown in FIGS. 5 and 6, some tumors wereinjected with beta-gal Ad. These tumors were harvested 24 hrs. later andfrozen tissue sections were assayed for beta-galactosidase expression.Tumors treated with p53 Ad for 2 or 6 weeks were still transduced bybeta-gal Ad, although transduction was lowest in the 468 tumors treatedfor 6 weeks with p53 Ad 5× per week. Sections from only 1 of the 3-468tumors injected in the 5× group had cells expressing beta-galactosidase.

Induction of Apoptosis In Vivo by p53 Ad

The MDA-MB-231 and MDA-MB-468 breast cancer xenografts in nude mice wereinjected with 1-5×10⁸ C.I.U. p53Ad or buffer 48 to 72 hrs. beforeharvest. The induction of apoptosis by p53 Ad was assayed using ApopTag®immunohistochemistry on tissue sections. Tumors injected with p53 Ad hadareas of extensive apoptosis along the needle track(s) of tumorsinjected intra-tumorally and around the outside border of tumorsinjected peritumorally. By contrast, tumors injected with buffer hadonly a few scattered apoptotic cells, as expected.

Comparison of Integrin Expression in MDA-MB-231 and MDA-MB-435 Cells

FACS analysis of integrin expression was performed on MDA-MB-231 andMDA-MB-435 cells to determine whether the low Ad transduction ofMDA-MB-435 cells was due to a deficiency in the alpha, integrins neededfor internalization of Ad types 2, 3, and 4 (Wickham et al. (1993) Cell,73: 309-319; Wickham et al. (1994) J. Cell Biol., 127: 257-264; andMathias et al. (1994) J. Virol. 68: 6811-6814). Both cell typesexpressed alpha_(v), alpha_(v)beta₃, alpha_(v)beta₅, and beta₁ integrinmoieties at approximately the same levels. Integrin alpha_(v)beta₃ andbeta₃ expression were higher on MDA-MB-435 cells than on MDA-MB-231cells.

Discussion: When a total dose of 2.2×10⁹ C.I.U. p53 Ad was administeredin 10 injections, tumor growth inhibition was 74% for MDA-MB-468 tumorsand 86% for MDA-MB-231 tumors, but was not significant for MDA-MB-435tumors. In MDA-MB-468 tumors, 61% of the total response wasp53-specific, while in MDA-MB-231 tumors, 43% of the total response wasp53-specific. The ability of beta-gal Ad to transduce MDA-MB-231,MDA-MB-468, and MDA-MB-435 cells in vitro was generally predictive ofthe in vivo results. At the same virus concentrations, −468 cells had aslightly higher transduction rate than MDA-MB-231 cells, whileMDA-MB-435 cells were resistant to adenovirus transduction. TheMDA-MB-435 results in vitro correlated with the very poor response invivo.

Systemic treatment of nude mice bearing MDA-MB-435 tumors, with ap53-liposome vector, has been shown to cause tumor growth inhibition,and in some cases, recession (Lesoon-Wood et al. (1995) Hum. Gene Ther.,6: 395-405). P53-liposome treatment also reduced the number of lungmetastases. These results demonstrate that the lack of MDA-MB-435 tumorresponse to p53 Ad-treatment in this study was not due to an inabilityof p53 to inhibit growth and metastasis of MDA-MB-435 tumors. Rather,these results suggest it was the low adenovirus transduction efficiencyof MDA-MB-435 cells that caused their nonresponsiveness to p53 Adtreatment.

The alpha_(v) integrins have been implicated as cellular elementsrequired for efficient internalization of type 2, 3, and 4 adenoviruses(Wickham (1993) supra.; Wickham (1994) supra.; and Mathias (1994)supra.). It is likely that alpha, integrins perform the same role fortype 5 Ad. Wickham et al. (1994) supra., observed 5-10-fold higherinternalization of a recombinant type 5 adenovirus in cells transfectedwith alpha_(v)beta₅ as compared to cells lacking alpha_(v) expression ortransfected with alpha_(v)beta₃. The human embryonic kidney −293 cellsused for production of the p53 Ad used herein express alpha_(v)beta₁,but not alpha_(v)beta₃ integrins (Bodary (1990) J. Biol. Chem. 265:5938-5941). Therefore, it seemed prudent to measure −435 cell expressionof the alpha_(v)beta₁, beta₃, and alpha₅ integrin subunits. BothMDA-MB-231 and MDA-MB-435 cells expressed roughly equivalent levels ofthe integrin family molecules. Therefore, the lack of Ad transduction ofMDA-MB-435 cells is not due to a deficiency in alpha₅ integrinexpression. Currently, no literature exists on the identity of thecellular receptor required for Ad binding to target cells. It ispossible that the MDA-MB-435 cells are deficient in this receptor orthat some other component required for viral binding, internalization,or gene expression is defective.

The continued efficacy of p53 Ad over multiple cycles of therapy wasexamined in the MDA-MB-231 and MDA-MB-468 tumor models. It appears thatefficacy decreased with continued dosing, however this effect needs tobe examined in more detail. Prevailing theory holds that adenovirusinfection generates a rapid inflammatory and cytolytic response mediatedby cytotoxic T cells in hosts with fully functional immune systems(reviewed by Wilson (1995) Nature Med. 4: 887-889). This T cell responseis stimulated by adenovirus antigens produced in host cells andpresented in conjunction with MHC moieties on the cell surface.Neutralizing antibodies specific for cells transduced by adenovirus areproduced later in the immune response and are believed responsible forthe reduced ability to reinfect host cells with adenovirus after theinitial inoculations. The athymic nude mice used in these studies have adefective T cell immune response to foreign antigens, but are able togenerate a B cell-mediated antibody response (Boven (1991) The NudeMouse in Oncology Research, Boston: CRC Press). The production ofneutralizing anti-adenovirus antibodies could explain the reducedefficacy of p53 adenovirus (p53 Ad) therapy over time in the presentstudies. The impaired immune function in nude mice and the poor bloodsupply to the interiors of the tumor xenografts could explain thepartial effectiveness of the p53 Ad even after 6 weeks of dosing and theability to infect a few tumor cells with beta-gal Ad even after repeatedp53 Ad injections.

In addition to breast cancer, a number of other cancers have beentreated with recombinant adenoviruses expressing wild-type p53. Thesereports include models of cervical cancer (Hamada (1996) Cancer Res. 56:3047-3054), prostate cancer (Eastham (1995) Cancer Res. 55: 5151-5155),head and neck cancer (Clayman (1995) Cancer Res. 55: 1-6), lung cancer(Wills (1994) supra.), ovarian cancer (13), glioblastoma (27, 28), andcolorectal cancer (13, 29). Collectively, these data support ongoingclinical investigations evaluating the effects of adenovirus-mediatedp53 gene therapy. The present results demonstrate the ability ofwild-type p53 to curtail cancerous cell growth in vivo in breast cancerxenografts expressing mutant p53. The present studies also confirm thatadenovirus appears to be an efficient delivery vehicle for the 53 whentarget cells express the appropriate viral “receptor(s)”.

Example 4 Further Investigations of Treatment Regimen on TumorInhibition

The invention provides for the treatment of various cancers by theadministration of nucleic acid expressing a tumor suppressor polypeptideusing various dosage regimens. The following example details theincreased efficacy of split dosing the administration of p53 expressingadenovirus of the invention.

In order to investigate the effect of a single dosage regimen ascompared to split doses administered over a period of time, scid miceinjected with MDA-MB-468 and MDA-MB-231 tumors were treated with a totaldose per mouse of 1×10⁹ I.U. p53 Ad (A/C/N/53) administered as a singlebolus injection or split into 3 or 5 injections administered once a dayover the course of a week (indicated by arrows in FIG. 7).

The results obtained with MDA-MB-468 tumors were similar to thoseobtained with MDA-MB-23 tumors and are illustrated in FIGS. 7 a, 7 b,and 7 c. In general, split dosing inhibited tumor growth better thansingle bolus injections with the 5 injection dosage regimen havingsignificant improvement over the 3 injection dosage regimen.

Example 5 Dexamethasone Mutes the Inhibition of Tumor Growth Associatedwith NK Cell-Mediated Anti-Adenovirus Immune Response

It has been demonstrated that repeated administration of adenovirusvectors can induce an anti-adenovirus immune response. To investigatewhether the immunosuppressant properties of low dose Dexamethasone (Dex)can inhibit the anti-adenovirus immune response (e.g., NK cell response)MDA-MB-231 tumors in scid mice were treated with recombinant virus ofthe invention in the absence and in the presence of dexamethasone.

Approximately 5×10⁶ MDA-MB-231 cells/mouse were injected into themammary fat pads of female scid mice on day 0. On day 11, dexamethasoneor placebo pellets were implanted subcutaneously. The 5 mg pellets weredesigned to release 83.3 μg dexamethasone/day continuously for 60 days(Innovative Research of America, Sarasota, Fla.). All mice received atotal of 10 peritumoral injections given once a day on days 14-18 and21-25 (0.1 ml per injection). The total virus dose was 2×10⁹C.I.U./mouse (p53 AD (A/C/N/53 or beta-galactosidase Ad). Treatmentswere as listed in Table 5. TABLE 5 Treatment of MDA-MB-231 tumors inscid mice. Group Hormone Gene Therapy 1 placebo buffer 2 placebobeta-gal Ad 3 placebo p53 Ad 4 dexamethasone buffer 5 dexamethasonebeta-gal Ad 6 dexamethasone p53 Ad

Low dosage dexamethasone treatment had no significant effect on thegrowth rate of MDA-MB-231 tumors in scid mice (p>0.05). No adverse sideeffects of dexamethasone were observed. Treatment of tumors withbeta-galactosidase adenovirus caused significant inhibition of tumorgrowth in placebo control tumors (p≦0.001, days 21-30), but not indexamethasone treated tumors (P>0.05, see FIG. 8). Tumors treated withplacebo and beta-gal Ad grew slower than tumors treated with placebo anddexamethasone (p≦0.01, days 23-30).

There was no significant p53-specific inhibition of tumor growth inplacebo control tumors (p>0.05). By contrast, tumors treated withdexamethasone and p53 Ad did grow significantly slower than tumorstreated with dexamethasone and beta-gal Ad (P≦0.02, days 21-30) orplacebo and p53 Ad (p≦0.04, days 21-30).

Low dose dexamethasone treatment thus muted the inhibition of tumorgrowth associated with the anti-adenovirus immune response (e.g., NKcell response) in scid mice without adverse side-effects. The data alsosuggest that low dose dexamethasone treatment may stimulate transgene(e.g., p53) expression driven by the CMV promoter in recombinantadenoviruses. Conversely, dexamethasone may increase adenovirustransduction efficiency and thereby increase tumor cell death.

The MDA-MB 231 breast cancer model was next used to evaluate theanti-tumor efficacy of Ad with and without p53 in mice with differingabilities to mount an immune response to foreign antigens. Nude micewith non-functional T cells, scid mice with nonfunctional T and B cellsbut elevated NK cells, and scid-beige mice with nonfunctional T, B, andNK cells were studied.

To study the efficacy of rAd5/p53 (described supra) against MDA-MD-231xenografts: nude mice were given a total dose of 2.2×10⁹ C.I.U. Ad permouse split into 10 injections on days 0 to 4 and 7 to 1. SCID mice weregiven a total virus dose=4×10⁹ C.I.U. split into 10 doses given on days0-4 and 7-11. SCID-Beige mice were given a total virus dose=1.6×10⁹C.I.U. split into 10 doses given on days 0-4 and 7-11. All mice weretreated with p53 Ad, beta-gal Ad, or vehicle alone.

In nude mice (non-functional T cells) or scid mice (nonfunctional T andB cells; elevated NK cells), intratumoral dosing with control Ad vector(no p53 insert) caused some inhibition of tumor growth. Ad expressingp53 (rAd5/p53) had greatly enhanced anti-tumor efficacy compared tocontrol Ad. By contrast, in scid-beige mice (nonfunctional T, B, and NKcells), anti-tumor efficacy was all due to p53 expression with no Advector component to the tumor growth inhibition. These data demonstratea previously unrecognized role for NK cells in Ad-mediated tumor growthinhibition. The data also suggest that suppression of the immune systemmight abrogate some vector-specific, NK cell mediated, side effects.

Example 6 Combination p53 Adenovirus and Chemotherapy Treatment

The invention provides for the combined administration of nucleic acidexpressing a tumor suppressor polypeptide and chemotherapeutic agents inthe treatment of neoplasms. The following example details the ability ofa p53 expressing adenovirus of the invention in combination with variousanti-cancer drugs, cisplatin, doxorubicin, 5-fluorouracil (5-FU),methotrexate, and etoposide, to treat neoplasms, and that thecombination therapy was more effective, i.e., was synergistic, atkilling tumor cells than either agent alone.

p53 Administered with Chemotherapeutic Drugs In Vitro

Cisplatin, Doxorubicin, 5 Fluorouracil (5-FU), Methotrexate, andEtoposide, in Combination with p53

The effect of the clinically-relevant anti-cancer drugs cisplatin,doxorubicin, 5-fluorouracil (5-FU), methotrexate, and etoposide, incombination with a tumor suppressor vector of the invention (A/C/N/53),was investigated in vitro. SCC-9 head and neck squamous cell carcinoma,SCC-15 head and neck squamous cell carcinoma, SCC-25 head and necksquamous cell carcinoma, and DU-145 prostate carcinoma cells weresubjected to one of three treatment regimes: In treatment 1, the cellswere pretreated with the anti-cancer chemotherapeutic twenty-four hoursbefore exposure to a p53 adenovirus construct A/C/N/53. In treatment 2,the cells were pretreated with the p53 adenovirus construct and thenlater contacted with the anti-cancer chemotherapeutic. In treatment 3,the cells were contacted simultaneously with both the anti-cancerchemotherapeutic and the p53 adenovirus.

All cell lines were obtained from ATCC (Rockville, Md.). SCC-9, SCC-15,and SCC-25 head and neck tumor cells (p53^(null)) were cultured in a 1:1mixture of DMEM and Ham's F-12 (GIBCO/Life Technologies, Grand Island,N.Y.) with 10% fetal calf serum (FCS; Hyclone, Logan, Utah), 0.4 μg/mlhydrocortisone (Sigma Chem. Co., St. Louis, Mo.), and 1% nonessentialamino acids (GIBCO) at 37° C. and 5% CO₂. SK-OV-3 human ovarian tumorcells (p53^(null)) and DU-145 human prostate tumor cells (p53^(mut) werecultured in Eagle's MEM plus 10% FCS at 37° C. and 5% CO₂. MDA-MB-231human mammary tumor cells (p53^(mut)) were cultured in DMEM (GE3CO) with10% fetal calf serum (Hyclone) at 37° C. and 5% CO₂.MDA-MB-468 humanmammary tumor cells (p53^(mut)) were cultured in Leibovitz's L-15 medium(GEBCO) containing 10% FCS at 37° C., no CO₂.

MDA-MB-231 mammary tumor cells carry an Arg-to-Lys mutation in codon 280of the p53 gene and express mutant p53 (Bartek (1990) supra). DU-145prostate tumor cells carry two p53 mutations on different chromosomes, aPro-to-Leu mutation in codon 223 and a Val-to-Phe mutation in codon 274(Isaacs (1991) Cancer Res. 51:4716-4720), and express mutant p53.SK-OV-3 ovarian tumor cells are p53-null (Yaginuma (1992) Cancer Res.52:4196-4199). SCC-9 cells have a deletion between codon 274 and 285resulting in a frame shift mutation; no immunoreactive p53 protein isdetectable in SCC-9 nuclei (Jung (1992) Cancer Res. 52:6390-6393;Caamano (1993) Am J. Pathol. 142:1131-1139; Min (1994) Eur. J. Cancer30B:338-345). SCC-15 cells have an insertion of 5 base pairs betweencodons 224 and 225; they produce low levels of p53 mRNA, but nodetectable p53 protein (Min (1994) supra). SCC-25 cells have loss ofheterozygosity (LOH) at chromosome 17 and a 2 base pair deletion incodon 209 on the remaining allele; p53 mRNA is not detectable in SCC-25cells and no immunoreactive p53 protein is observed in their nuclei(Caamano (1993) supra). Approximately 1.5×10⁴ cells in culture medium(as described in example 1) were added to each well on a 96 wellmicrotitre plate and cultured for about 4 hours at 37° C. and 5% CO₂.

Construction and propagation of the human wild-type p53 and E. coligalactosidase (beta-gal) adenoviruses (Ad) have been describedpreviously (Wills (1994) supra). The concentration of infectious viralparticles was determined by measuring the concentration of viral hexonprotein positive 293 cells after a 48 hr. infection period (Huyghe(1995) supra). C.I.U. is defined as cellular infectious units.p53-expressing adenoviruses were administered in phosphate buffer (20 mMNaH₂PO₄, pH 8.0, 130 mM NaCl, 2 mM MgCl₂, 2% sucrose). The drug, the p53adenovirus, or the appropriate vehicle/buffer was added to each well.For in vitro studies with p53 Ad, cells were plated at a density of1.5×10⁴ cells/well on a 96-well plate and cultured for 4 hrs. at 37° C.and 5% CO₂. Drug, p53 Ad, or the appropriate vehicle was added to eachwell and cell culture was continued overnight. Then drug, p53 Ad, or theappropriate vehicle was added to each well. Cell culture was continuedfor an additional 2 days.

Cell death was then quantitated according to the MTT assay as describedby Mosmann (1983) J. Immunol. Meth., 65: 55-63. Briefly, approximately25 μl of 5 mg/ml MTT vital dye [3-(4,5dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] was added to eachwell and allowed to incubate for 3-4 hrs. at 37° C. and 5% CO₂. Then 100μl of 10% SDS detergent was added to each well and allowed to incubateovernight at 37° C. and 5% CO₂. Signal in each well was then quantitatedusing a Molecular devices microtiter plate reader.

In all cases, using cisplatin (see Table 6 for summary results),doxorubicin (see Table 7 for summary results), 5-FU, methotrexate, andetoposide, the combination therapy was more effective at killing tumorcells than either agent alone. The combination of methotrexate and p53Ad was tested in one cell line. When SCC-15 cells were treated with 0.7μM methotrexate 24 hours before 5 m.o.i. p53 Ad, the combinedantiproliferative effect of the two drugs was only 5% more than with p53Ad, alone, although this difference was statistically significant(p≦0.003). Pretreatment of DU-145 cells with 2.6 μM etoposide 24 hoursbefore 5 or 10 m.o.i. p53 Ad resulted in greater combined efficacy overeither drug alone (p≦0.0001). When SCC-15 cells were treated with 0.3 μMetoposide 24 hours before 5 m.o.i. p53 Ad, the combinedantiproliferative effect of the two drugs was only 5% more than with p53Ad alone, although this difference was also statistically significant(ps0.003). The combination of tumor suppressor gene therapy andanti-neoplastic agents did not demonstrate antagonistic effects.

In a second experiment, the efficacy of treatment of normal cells (MRC-9cells) was compared with tumor cells (FIG. 9). In this experiment,growth was assayed as ³H-thymidine incorporation rather than the MTTassay. The normal cells (diploid fibroblast MRC-9 cells) did not showmore pronounced effects with combination treatment. As would beexpected, the effect of tumor suppressor alone was negligible in normalcells and highly significant in tumor cells. In contrast, theanti-cancer chemotherapeutic alone (e.g., cisplatin, doxorubicin, 5-FU,methotrexate, and etoposide) was more effective in normal cells thancancer cells (see FIG. 9). TABLE 6 Anti-proliferative effects of p53 Adin combination with cisplatin. Greater Combined Efficacy? Cell Line p53Protein Tissue Type Cisplatin First p53 Ad First Simultaneous SK-OV-3Null Ovarian Yes (p ≦ 0.0001) Yes (p ≦ 0.0001) Yes (p ≦ 0.0001) SCC-9Null Head & Neck Yes (p ≦ 0.0001) Yes (p ≦ 0.0001) Yes (p ≦ 0.0001)SCC-15 Null Head & Neck Yes (p ≦ 0.0001) ND ND SCC-25 Null Head & NeckYes (p ≦ 0.0001) Yes (p ≦ 0.0001) Yes (p ≦ 0.0001) MDA-MB-468 MutantBreast Yes (p ≦ 0.0001) ND Yes (p ≦ 0.0001) MDA-MB 231 Mutant Breast Yes(p ≦ 0.0002) Yes (p ≦ 0.0001) Yes (p ≦ 0.0001)ND = not done

TABLE 7 Anti-proliferative effects of p53 Ad in combination withdoxorubicin. Greater Combined Efficacy? Cell Line p53 Protein TissueType Dox First p53 Ad First Simultaneous SK-OV-3 Null Ovarian Yes (p ≦0.0001) Yes ((p ≦ 0.0001) Yes (p ≦ 0.0001) SCC-9 Null Head & Neck Yes (p≦ 0.0001) Yes (p ≦ 0.0001) Yes (p ≦ 0.0001) SCC-15 Null Head & Neck Yes(p ≦ 0.0001) Yes (p ≦ 0.0001) Yes (p ≦ 0.0001) SCC-25 Null Head & NeckYes (p ≦ 0.0001) Yes (p ≦ 0.0001) Yes (p ≦ 0.0001) DU-145 MutantProstate Yes (p ≦ 0.0001) Yes (p ≦ 0.0001) Yes (p ≦ 0.0001) MB-231Mutant Breast Yes (p ≦ 0.0001) Yes (p ≦ 0.0001) Yes (p ≦ 0.0001)

Doxorubicin and p53 Effects on Human Hepatocellular Carcinoma

The following example details the ability of a p53 expressing adenovirusof the invention in combination with doxorubicin to treat neoplasms, andthat the combination therapy was more effective, i.e., was synergistic,at killing tumor cells than either agent alone. The results demonstratea synergistic interaction between the p53 expressing vector of theinvention (ACN53) and doxorubicin.

Doxorubicin (Adriamycin) and p53 (ACN53, the recombinant adenovirusconstruct expressing the human wild-type p53 transgene) wereadministered to the following cell lines: HLE, a human hepatocellularcarcinoma cell line (Hsu (1993) Carcinogenesis 14:987-992; Farshid(1992) J. Med. Virol. 38:235-239; Dor (1975) Gann. 66:385-392), with amutated p53; HLF, a human hepatocellular carcinoma cell line with amutated p53 (ibid); Hep 3B, a human hepatocellular carcinoma with a nullp53 (Hasegawa (1995) In vitro Cell Dev Biol Anim. 31:55-61); Hep G2, ahuman hepatocellular carcinoma with p53 wild-type (ibid); and, SK-HEP-1,a human liver adenocarcinoma with p53 wild-type (Lee (1995) FEBS Lett.368:348-352). Cell viability measured with the live cell probe calcienAM (Molecular Probes) (see, e.g., Poole (1993) J. Cell Sci.106:685-691). The substrate calcien AM is cleaved by cellular esterasesto generate a fluorescent product.

Cells were plated in 96 well plates (5×10³ cells/well), allowed toadhere overnight, treated in triplicate with dilutions of ACN53 anddilutions of doxorubicin on day 0, such that a dose response-curve fordoxorubicin treatment was generated with each dose of ACN53. On day 3media was aspirated and calcien AM in PBS was added to the cells.Fluorescent intensity of each well was determined using fluorescentplate reader. Fluorescent value from wells with no cells were subtractedand data was expressed as the percent viability (fluorescent intensity)compared to untreated control wells. ED₅₀ values were used to generateisobologram plots to assess the interaction between ACN53 anddoxorubicin.

Isobologram analysis for each cell line showed a synergistic interactionbetween the p53 expressing vector of the invention (ACN53) anddoxorubicin; this synergy was independent of the p53 status of the cellline. Note, however, the ED₅₀ for ACN53 in the absence of doxorubicin ishigher in the p53 wild-type cell lines than in the p53 altered lines.

In another similar experiment, HLE cells were plated in 96 well plates(5×10³ cells/well); allowed to adhere overnight; and, treated intriplicate with dilutions of ACN53 and dilutions of doxorubicin suchthat a dose response curve for doxorubicin treatment was generated witheach dose of ACN53. Three groups were used to test the effects on dosingorder on the interaction between ACN53 and doxorubicin. group day 0 day1 day 2 day 3 simultaneous ACN53, doxorubicin harvest ACN53 first ACN53doxorubicin harvest doxorubicin first doxorubicin ACN 53 harvest

Cells were incubated for 3 days after first treatment. Media wasaspirated and calcien AM in PBS was added to the cells. Fluorescentintensity of each well was determined using fluorescent plate reader.Fluorescent value from wells with no cells were subtracted and data wasexpressed as the percent viability (fluorescent intensity) compared tountreated control wells. ED₅₀ values were used to generate isobologramplots to assess the interaction between ACN53 and doxorubicin.Isobologram analysis for each dosing regimen showed similar interaction,consistent with synergy, between ACN53 and doxorubicin in HLE cells thatwas independent of the dosing order of the treatment.

p53 With Chemotherapeutic Drugs In Vivo

The effect of the clinically-relevant anti-cancer drugs cisplatin,doxorubicin, and 5-fluorouracil (5-FU), in combination with a tumorsuppressor vector of the invention (A/C/N/53), was further investigatedin vivo.

C.B. 17/ICR-scid mice were purchased from Taconic Farms (Germantown,N.Y.) or Charles River Laboratories (Wilmington, Mass.). Athymic nu/numice were purchased from Charles River Laboratories. All mice weremaintained in a VAF-barrier facility and all animal procedures wereperformed in accordance with the rules set forth in the N.I.H. Guide forthe Care and Use of Laboratory Animals. Tumor volumes for differenttreatment groups on each day were compared by Student's t test usingStatview II software (Abacus Concepts, Berkeley, Calif.). Tumor growthcurves were constructed to show mean tumor volume±s.e.m. There werenormally ten-mice per group.

SK-OV-3 Ovarian Tumor Model:

Established intraperitoneal SK-OV-3 tumors were treated withintraperitoneal doses of vehicles, p53 Ad, cisplatin, or both drugs.Mice were given six injections of p53 Ad over a period of two weeks. Thetotal virus dose was 1.5×10⁹ C.I.U. (3.1×10¹⁰ viral particles).

Cisplatin efficacy: Female scid mice were injected with 5×10⁶ SK-OV-3ovarian tumor cells, I.P., on day 0. Mice were dosed I.P. on days 6, 8,10, 13, 15, and 17 (p53 Ad only on D17). Mice received 0.2 ml totalvolume (0.1 ml cisplatin vehicle or cisplatin plus 0.1 ml Ad buffer orp53 Ad). The p53 Ad dose was 2.5×10⁸ C.I.U./mouse/day (5.2×10⁹ viralparticles). The cisplatin dose was 2 mg/kg/day. Tumors were harvestedand weighed on day 20.

Mice in one treatment group received five doses of cisplatinsimultaneously with the first five p53 Ad doses. This dose ofintraperitoneal p53 Ad reduced mouse tumor burden only 17% by day 20(p≦0.01). However, when combined with cisplatin, p53 Ad caused a 38%decrease in tumor burden as compared to cisplatin alone (p≦0.0008). Micetreated with drug vehicles or with p53 Ad alone had bloody ascites andinvasive tumor nodules in the diaphragm muscle. These symptoms wereabsent in the mice treated with cisplatin alone or cisplatin with p53Ad.

Cisplatin/Paclitaxel efficacy: Female scid mice were injected with2.5×10⁶ SK OV-3 ovarian tumor cells, I.P., on day 0. Mice were dosedI.P. on days 7, 9, 11, 16, and 18. Mice received 0.3 ml total volume(0.1 ml cisplatin vehicle or cisplatin plus 0.1 ml paclitaxel vehicle orpaclitaxel plus 0.1 ml Ad buffer or p53 Ad). The p53 Ad dose was 2.5×10⁸C.I.U./mouse/day (5.2×10⁹ viral particles). The cisplatin dose was 0.5mg/kg/day. The paclitaxel dose was 1 mg/kg/day. Tumors were harvestedand weighed on day 30. n=7=10 mice per group.

In this second study, SK OV-3 ovarian tumors were treated withintraperitoneal doses of vehicles, p53 Ad, cisplatin plus paclitaxel, orall three drugs simultaneously. The combination of all three drugsreduced tumor burden 34% more than the combination of cisplatin pluspaclitaxel, demonstrating the enhanced efficacy of the three drugcombination (p≦0.0006).

DU-145 Prostate Tumor Model:

Cisplatin Efficacy:

Intraperitoneal DU-145 tumors were treated with intraperitoneal doses ofvehicles, p53 Ad, cisplatin, or both drugs. Male scid mice were injectedwith 2.5×10⁶ DU-145 cells, I.P., on day 0. Mice were dosed I.P. on days7, 9, 11, 14, and 16. Mice received 0.2 ml total volume (0.1 mlcisplatin vehicle or cisplatin plus 0.1 ml Ad buffer or p53 Ad). The p53Ad dose was 83×10⁸ C.I.U./mouse/day (2.9×10¹⁰ PN). The cisplatin dosewas 1 mg/kg/day. Tumors were harvested and weighed on day 22. Thecombination of p53 Ad and cisplatin had greatly enhanced anti-tumorefficacy compared to either drug alone (p≦0.0004).

MDA-MBA68 Mammary Tum r Model:

Cisplatin Efficacy:

Established MDA-MBA68 tumors were treated with vehicles, p53 Ad,cisplatin, or both drugs. Female scid mice were. injected with 1×10⁷MDA-MB-468 cells into the mammary fat pad, 11 days before the start ofdosing on day 0. The intraperitoneal cisplatin dose was 1 mg/kg/day. Theintratumoral p53 Ad dose was 8.3×10⁸ CIU/mouse/day (2.9×10¹⁰ viralparticles) given simultaneously on days 0-4. p53 Ad had enhancedefficacy when combined with cisplatin (days 8-31, p≦0.0004).

Doxorubicin Efficacy:

In a second experiment, MDA-MB-468 tumors were treated with vehicles,p53 Ad, doxorubicin, or both drugs. Female nude mice were injected with1×10⁷ MDA-MB-468 cells subcutaneously 12 days prior to the start ofdosing on day 0. The intraperitoneal doxorubicin dose was 4 mg/kg/day ondays 0, 2, 7, and 9. The intratumoral p53 Ad dose was 5×10⁸CIU/mouse/day (1.03×10¹⁰ viral particles) on days 0-4 and 7-11.

p53 Ad had greater efficacy when administered in combination withdoxorubicin (days 14-24, p≦0.05).

SCC-15 Head and Neck Tumor Model:

5-Fluorouracil Efficacy:

Subcutaneous SCC-15 tumors were treated with vehicles, p53 Ad,5-fluorouracil (5-FU), or both drugs. Scid mice were injected with 5×10⁶SCC-15 cells subcutaneously 7 days prior to the start of dosing on day0. The intraperitoneal 5-fluorouracil dose was 50 mg/kg/day in 40%hydroxypropyl-beta-cyclodextran (Cerestar Inc., Hammond, Ind.) givenI.P. on days 0, 7, and 14 (once a week for 3 weeks). The p53 Ad dose was2×10⁸ CIU/mouse/day (4×10⁹ viral particles), on days 0, 1, 7, 8, 14, and15 (6 intratumoral injections over a period of 3 weeks). The 5-FU doseof 50 mg/kg was given. The combination of p53 Ad and 5-FU resulted ingreater antitumor activity than, when either drug was used alone(p≦0.04).

p53 With FPT Inhibitor

The effect of a farnesyl protein transferase inhibitor in combinationwith a tumor suppressor vector designated AICIN/53 was investigated invitro. The following examples detail the ability of a p53 expressingadenovirus of the invention in combination with the FPT inhibitordesignated “FPT39,” as described in International Application WO97/23478, filed Dec. 19, 1996, where FPT39 is designated compound“39.0,” see pg 95 of WO 97/23478) to treat neoplasms, and that forcombination therapy against prostate tumor cells and mammary tumor cellswas more effective at killing tumor cells than either agent alone.

Anti-Proliferative Efficacy of rAd5/p53 and FPT39 Against SK-OV-3Ovarian Tumor

Methods: SK-OV-3 human ovarian tumor cells (p53^(mull)) were aliquotedinto 96-well plates at a density of 250 cells per well in Eagle's MEMplus 10% fetal bovine serum. Cells were then incubated at 37° C. and 5%CO₂ for 4 hours. FPT39 or drug vehicle was added to each well and cellculture was continued for 3 days. After 3 days, untreated cells in somewells were counted in order to calculate the amount of rAd5/p53 to add.Then rAd5/p53 or drug vehicle was added to each well and cell culturewas continued for another 3 days. Cell proliferation was measured usingthe MTT assay. Briefly, 25 ul of 5 mg/ml MTT vital dye [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to eachwell and allowed to incubate for 3-4 hrs. at 37° C. and 5% CO₂. Then,100 ul of 10% SDS detergent was added to each well and the incubationwas continued overnight. Fluorescence in each well was quantitated usinga Molecular Devices microtiter plate reader. Cell proliferation data wasanalyzed using the Thin Plate Spline statistical methodology ofO'Connell and Wolfinger (1997) J. Comp. Graph. Stat. 6: 224-241.

Results: rAd5/p53 and FPT39 had additive efficacy in inhibiting cellgrowth. Neither synergism (p>0.05) nor antagonism (p>0.05) weredemonstrated in this experiment.

Anti-Proliferative and Synergistic Efficacy of rAd5/p53 and FPT39 (FPTInhibitor) Against DU-145 Prostate Tumor Cells

Methods: DU-145 human prostate tumor cells (p53^(mut)) were treated withFPT39 or drug vehicle, and rAd5/p53, and the cell cultures weresubsequently analyzed, as described above for the SK-OV-3 human ovariantumor cells. The experiment was repeated twice.

Results: Experiment 1: rAd5/p53 and FPT39 had additive efficacy ininhibiting cell growth. Neither synergism (p>0.05) nor antagonism(p>0.05) were demonstrated in this experiment.

Experiment 2: rAd5/p53 and FPT39 had synergistic efficacy (p=0.0192).These results demonstrate that rAd5/p53 and FPT39 can interact and havesynergistic efficacy against prostate tumor cell proliferation.

Anti-Proliferative and Synergistic Efficacy of rAd5/p53 and FPT39 (FPTInhibitor) Against MDA-MB-231 Mammary Tumor Cells

Methods: MDA-MB-231 human breast cancer cells (p53^(mut)) were treatedwith FPT39 or drug vehicle, and rAd5/p53, and the cell cultures weresubsequently analyzed, as described above for the SK-OV-3 human ovariantumor cells. The experiment was repeated twice.

Results: Experiment 1: rAd5/p53 and FPT39 had additive efficacy. Neithersynergism (p>0.05) were demonstrated in this experiment.

Experiment 2: rAd5/p53 and FPT39 had additive efficacy over most of theresponse surface. However, synergism was evident at isoboles greaterthat or equal to 70 (i.e., less than 30% of cells killed, p=0.0001).These results demonstrate that rAd5/p53 and FPT39 can interact and havesynergistic efficacy against human breast cancer cell proliferation.

Example 7 Immune Response Profile in Patients with Metastatic HepaticCarcinomas Treated with Adenovirus Vector Carrying p53

The invention provides for the combined in vivo administration ofnucleic acid expressing p53 and other chemotherapeutic agents in thetreatment of neoplasms. The following example details the ability of thep53 expressing adenovirus of the invention to increase the levels oftumor-killing lymphocytes found within a human liver carcinoma.

The aim of this study was to characterize the genotypes and phenotypesof the tumor infiltrating lymphocytes (TILs) in metastatic livercarcinomas from the colon harboring p53 mutations (for discussion TILs,see, e.g., Wang (1997) Mol. Med. 3:716-731; Marrogi (1997) Int. J.Cancer 74:492-501). A total of 16 patients were treated in a doseescalating manner, 10⁹-10¹¹ particles, through hepatic arterycanalization with an adenovirus vector carrying wild type p53 gene. Atotal of four biopsies from each patient was obtained 3 to 7 days afteradministering the adenoviral vector. Immunohistochemical analysis wereperformed on the frozen tissues obtained from normal liver andtumor-host tissue interface sites. Computer assisted image analysis wasperformed to quantitate immunoreactivity to the following monoclonalantibodies: CD3, CD4, CD8, CD25, CD28, CD56, HLA-DR, IFN-gamma,TNF-alpha and IL-2. An increase in TILs (CD3⁺ and CD4⁺) population wasobserved with the maximum at 7.5×10¹⁰ particles. At higher doses, adecrease in CD3⁺ and CD4⁺ population was observed. An inversecorrelation was observed for CD8⁺ cells. At the highest dose (2.5×10¹¹)an increase in the CD3⁺, CD4⁺ and CD8⁺ population was observed in thetumor as compared to the normal. These results demonstrate that deliveryof high doses of adenovirus particles results in increased TILs composedof CD4⁺ and CD8⁺ population.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. A kit for the treatment of mammalian cancer or hyperproliferativecells, said kit comprising: a first container comprising a tumorsuppressor nucleic acid selected from the group consisting of wild-typep53 nucleic acid, or a retinoblastoma (RB) nucleic acid; and a secondcontainer comprising at least one anti-mitotic agent; wherein themammalian cancer cells or hyperproliferative cells comprise a memberselected from the group consisting of a human head and neck cell, anovarian cell, a prostate cell, and a breast cell.
 2. The kit of claim 1,wherein said tumor suppressor nucleic acid encodes a wild-type p53protein.
 3. The kit of claim 1, wherein said anti-mitotic agent ispaclitaxel or a paclitaxel derivative.
 4. The kit of claim 1, furthercomprising instructions describing the administration of both said tumorsuppressor nucleic acid and said anti-mitotic agent to inhibit thegrowth or proliferation of said cells.
 5. The kit of claim 1, whereinsaid tumor suppressor nucleic acid is selected from the group consistingof p53, p110^(RB), and p56^(RB).
 6. The kit of claim 1, wherein saidfirst container contains a nucleic acid that is contained in arecombinant adenoviral vector.
 7. The kit of claim 6, wherein saidnucleic acid is contained in a recombinant adenoviral vector comprisinga partial or total deletion of a protein IX DNA and comprising a nucleicacid encoding a p53 protein.
 8. The kit of claim 7, wherein saiddeletion of the protein IX gene sequence extends from about 3500 bp fromthe 5′ viral terminus to about 4000 bp from the 5′ viral terminus. 9.The kit of claim 8, wherein the adenoviral vector further comprises adeletion of DNA sequence designated E1a and E1b.
 10. The kit of claim 6,wherein said recombinant adenoviral vector comprises the adenovirus type2 major late promoter or the human CMV promoter, the adenovirus type 2tripartite leader cDNA and a human p53 cDNA.
 11. The kit of claim 6,wherein said vector is A/C/N/53.
 12. A pharmacological composition fortreating mammalian cancer cells or hyperproliferative cells, comprisinga tumor suppressor nucleic acid and at least one anti-mitotic agent,wherein the mammalian cancer cells or hyperproliferative cells comprisea member selected from the group consisting of a human head and neckcell, an ovarian cell, a prostate cell, and a breast cell.
 13. Thecomposition of claim 12, wherein said anti-mitotic agent is paclitaxelor a paclitaxel derivative.
 14. The composition of claim 12, whereinsaid tumor suppressor nucleic acid is selected from the group consistingof a nucleic acid that encodes a wild-type p53 protein, and a nucleicacid that encodes a retinoblastoma (RB) protein.
 15. The composition ofclaim 12, wherein said nucleic acid encodes a wild-type p53 protein. 16.The composition of claim 12, wherein said nucleic acid encodes a saidretinoblastoma p110^(RB) or a p56^(RB).
 17. The composition of claim 12,wherein said nucleic acid is contained in recombinant adenoviral vector.18. The composition of claim 17, wherein said nucleic acid is containedin a recombinant adenoviral vector comprising a partial or totaldeletion of a protein IX DNA and comprising a nucleic acid encoding aP53 protein.
 19. The composition of claim 18, wherein said deletion ofthe protein IX gene sequence extends from about 3500 bp from the 5′viral terminus to about 4000 bp from the 5′ viral terminus.
 20. Thecomposition of claim 19, wherein the adenoviral vector further comprisesa deletion of DNA sequence designated E1a and E1b.
 21. The compositionof claim 17, wherein said recombinant adenoviral vector comprises theadenovirus type 2 major late promoter or the human CMV promoter, theadenovirus type 2 tripartite leader cDNA and a human p53 cDNA.
 22. Thecomposition of claim 17, wherein said vector is A/C/N/53.
 23. Thecomposition of claim 13, wherein said paclitaxel or paclitaxelderivative is paclitaxel.
 24. A composition comprising a mammaliancancer or hyperproliferative cell, wherein said cell contains anexogenous tumor suppressor nucleic acid; and paclitaxel or a paclitaxelderivative.
 25. The composition of claim 24, wherein said tumorsuppressor nucleic acid is a nucleic acid that encodes a tumorsuppressor protein selected from the group consisting of wild-type p53protein, and a retinoblastoma (RB) protein.
 26. The composition of claim24, wherein said tumor suppressor nucleic acid encodes a wild-type p53protein.
 27. The composition of claim 25, wherein said retinoblastomaprotein is a p110^(RB) or a p56^(RB).
 28. The composition of claim 24,wherein said cell is present in a mammal.
 29. The composition of claim24, wherein said cell comprises a neoplastic cell.
 30. The compositionof claim 29, wherein said neoplastic cell comprises a cancer cellselected from the group consisting of an ovarian cancer cell, apancreatic cancer cell, a non-small cell lung cancer cell, a small celllung cancer cell, a hepatocarcinoma cell, a melanoma cell, aretinoblastoma cell, a breast tumor cell, a colorectal carcinoma cell, aleukemia cell, a lymphoma cell, a brain tumor cell, a cervical carcinomacell, a sarcoma cell, a prostate tumor cell, a bladder tumor cell, atumor cell of the reticuloendothelial tissues, a Wilm's tumor cell, anastrocytoma cell, a glioblastoma cell, a neuroblastoma cell, anosteosarcoma cell, a renal cancer cell, and a head and neck cancer cell.31. The composition of claim 24, wherein the mammalian cancer cell orhyperproliferative cell comprises a member selected from the groupconsisting of a human head and neck cell, an ovarian cell, a prostatecell, and a breast cell.
 32. The composition of claim 24, wherein themammalian cancer or hyperproliferative cell is deficient in endogenousp53 tumor suppressor nucleic acid.